The immune system in disease: gestational trophoblastic tumours. Trophoblastic tumours form a spectrum of disease from the borderline malignancy of HM to highly aggressive choriocarcinoma. Their management requires the integration of the information derived from serial hCG estimations, the clinical history and pattern of spread of the disease, so that our understanding of the prognostic variables can be applied appropriately. This maximizes the patient's chances of complete remission from her disease with the minimum of toxicity. Given our knowledge of this group of diseases and an integrated approach to management, it should be uncommon for any woman to die from her trophoblastic tumour.


The tissue-implant interface during degradation of absorbable polyglycolide fracture fixation screws in the rabbit femur. A transverse transcondylar osteotomy of the distal femur was fixed with an axially placed absorbable fracture fixation screw made of polyglycolide (PGA) in 25 rabbits. Changes at the tissue-implant interface accompanying degradation of the screw were examined histologically, histomorphometrically, and microradiographically seven, 20, 40, 80, and 250 days after implantation. At seven days postimplantation, a layer of fibroblasts was seen surrounding the implant, and new bone formation was discernible in the host tissues adjacent to this membranous structure. At 20 days postimplantation, the geometry of the screw was still intact and the tissue-implant boundary was distinct. The first signs of invasion of vascular granulation tissue into the implant were observed 40 days after implantation, at which time the osteotomies were united. The apparent walling-off response by formation of new trabecular bone outlining the PGA profile continued, with the greatest mean trabecular bone volume fraction at the interface, 23.9%, measured at 40 days. A significant decrease in the new bone volume occurred between 40 and 80 days postimplantation. The intensity of the foreign-body reaction seen was histologically moderate. The giant cell count was highest at 80 days postimplantation, when the migratory activity of phagocytic cells had transported intracellular particulate polymeric debris 400-800 microns away from the original tissue-implant boundary. At 250 days postimplantation, no birefringent polymeric material could be seen in the specimens. No contraindications for the clinical application of PGA implants emerged in this study.


Preexisting cardiopulmonary disease attenuating the atrial natriuretic peptide response. Results in patients with acute respiratory failure. The purpose of this study was to evaluate the pathophysiologic role of atrial natriuretic peptide (ANP) as a pulmonary artery vasodilator in patients with acute respiratory failure receiving artificial ventilation. Twenty-one consecutive patients were studied, 12 without and 9 with preexisting cardiopulmonary disease. Pulmonary artery plasma ANP levels were significantly higher than the levels obtained in the superior vena cava and radial artery. Plasma ANP levels correlated significantly with the plasma levels of its second messenger, guanosine 3',5'-cyclic monophosphate (cGMP). In the 12 patients without prior cardiopulmonary disease, plasma ANP levels correlated significantly with mean pulmonary arterial pressure (MPAP). This correlation was not found in the nine patients with preexisting cardiopulmonary disease. The cGMP/ANP ratio, indicating the biologic effect of ANP, was also higher in the patients without preexisting cardiopulmonary disease. These results are compatible with clearance and vasodilator activity of ANP in the pulmonary vascular bed, but only in patients without preexisting cardiopulmonary disease.


Host defense activity in various hosts. Human neutrophil NADPH oxidase activity. The superoxide generation of neutrophil NADPH oxidase from healthy subjects, patients with respiratory infections, and patients receiving effective therapy with antibiotics or steroids was investigated. In young healthy nonsmokers the mean oxidase activity of neutrophils in women was significantly lower than that in men. In healthy women the mean oxidase activity was significantly lower in young nonsmokers than in young smokers or the elderly. In young nonsmokers, oxidase activity significantly increased during respiratory infections; however, in elderly nonsmokers, no significant increase in oxidase activity was observed during respiratory infections. The mean oxidase activity in patients receiving steroids was very low. In in vitro experiments using cell-free activation systems of NADPH oxidase, steroids were found to injure the membrane-bound components of the oxidase enzyme. These results suggest that decreased superoxide generation in patients receiving steroids may result from steroid-induced damage in the membrane-bound components of the NADPH oxidase system. The inhibitory effect of steroids on superoxide production may reduce bactericidal action of neutrophils, ie, one defense mechanism of the body against many kinds of pathogens. Therefore, long-term therapy with steroids in the elderly should be avoided at all costs.


Obese Zucker (fa/fa) rats exhibit normal target sensitivity to corticosterone and increased drive to adrenocorticotropin during the diurnal trough. Genetically obese Zucker (fa/fa) rats exhibit numerous metabolic and endocrine disorders associated with modest hypercorticosteronemia and reported changes in peripheral target tissue sensitivity to glucocorticoids. In this study we investigated phenotypic differences in basal and stress-induced ACTH and corticosterone (B) secretion in intact and adrenalectomized lean and obese male Zucker rats. In addition, we determined whether differences in the sensitivity to B of plasma ACTH and insulin secretion as well as other peripheral B targets could be observed between the two phenotypes. There were no significant differences in basal ACTH or B in either the morning (AM) or evening (PM) in intact obese and lean rats; however, mean B was increased in the obese rats in the AM, and signs of chronically increased adrenocortical activity were observed, including increased adrenal weight and intraadrenal phenylethanolamine-N-methyl transferase activity and decreased thymus weight. In a second experiment, B was significantly elevated 3 min after either administration in obese compared to lean rats; however, there was no significant difference in B between the groups at 10 min, nor were ACTH levels at these times different. Five days after adrenalectomy with sc B replacement, ACTH was decreased as a function of B in both phenotypes under AM basal and stress conditions. The IC50 values for inhibition of basal ACTH by B were 3.16 and 4.17 micrograms/dl in lean and obese rats, respectively. Under stress conditions, the IC50 values were not different (4.39 micrograms/dl for lean and 4.24 micrograms/dl for obese rats). B dose-dependent increases in body and epididymal fat depot weights were greater in obese than in lean rats, an expected result because of elevated insulin levels in this group. Insulin exhibited only small B-dependent increases, and thymus weight decreased in a B-dependent fashion; there were no differences in the sensitivity to B of these measures between lean and obese rats. We conclude that 1) there is no evidence for altered sensitivity to B in obese rats for any of the B-sensitive end points measured; and 2) basal adrenocortical activity is slightly elevated, and the sensitivity of ACTH to B feedback is decreased in obese rats under AM conditions in the absence of external stress.(ABSTRACT TRUNCATED AT 400 WORDS)


Neurointermediate lobe peptides recruit prolactin-secreting cells exclusively within the central region of the adenohypophysis. There is strong evidence that the hypophyseal neurointermediate lobe (NIL) mediates 17 beta-estradiol (E2)-induced PRL secretion in rats. Our laboratory has previously demonstrated that E2 stimulates NIL cells to release an activity that acutely increases the relative abundance of PRL-releasing cells in anterior pituitary (AP) cell cultures. We later found that secretory products of the NIL melanotropes, specifically the acetylated forms of alpha MSH and beta-endorphin (beta END), can account for this activity. Given that blood from the NIL initially perfuses the region of the AP proximal to the NIL, we tested the hypothesis that this specific area was preferentially responsive to the lactotrope recruitment activities. AP glands from ovariectomized rats were dissected into an inner zone, proximal to the NIL, and the remaining outer zone of the gland, then dispersed with trypsin. The resulting cells were cultured for 16 h, either alone or in coculture with NIL cells, and subsequently treated with medium alone (control) or with alpha MSH, beta END, or E2 (all at 1 x 10(-7) M) for 3 h and then subjected to reverse hemolytic plaque assays for PRL release. Under control conditions, the proportion of PRL-secreting cells was significantly greater in cultures from the outer zone of the AP than in those from the corresponding inner zone of the gland. Treatment of AP cells from the inner zone with alpha MSH, beta END, or the E2-induced NIL activity significantly increased the percentage of PRL secretors by about 8% of all AP cells. In contrast, the fraction of PRL-secreting cells in cultures from the outer zone was not affected by these treatments. We conclude that the recruitment of PRL-secreting cells in response to products of the NIL occurs only in that region of the AP proximal to the NIL.


Corticotropin-releasing hormone and adrenocorticotropic hormone concentrations in cerebrospinal fluid of dogs with pituitary-dependent hyperadrenocorticism. There is still some controversy concerning the question of whether Cushing's disease in man is caused by a primary dysfunction of the pituitary or a hypothalamic disorder. In the latter option, excessive hypothalamic stimulation of pituitary corticotropes would cause or contribute to the genesis of POMC-secreting adenomas. In the present study cerebrospinal fluid (CSF) CRH levels and levels of ACTH and cortisol in CSF and plasma were measured in clinically healthy dogs, in dogs with pituitary-dependent hyperadrenocorticism (PDH), and in dogs with hyperadrenocorticism due to an adrenocortical tumor (ATH). In CSF from dogs with PDH, CRH concentrations (226.6 +/- 14.4 ng/liter) were significantly (P < 0.05) lower than those in control dogs (309.5 +/- 20.3 ng/liter). In the dogs with ATH, CSF CRH concentrations (211.0 +/- 40.3 ng/liter) were in the range of those in PDH dogs. In dogs with ATH, CSF ACTH levels (13.0 +/- 3.0 ng/liter) were significantly (P < 0.05) lower than those in control dogs (63.4 +/- 3.5 ng/liter), whereas in dogs with PDH, the levels (116.8 +/- 47.5 ng/liter) were not different from those in the control group. In control dogs, the concentrations of CSF CRH and plasma ACTH were significantly correlated (r = 0.635; P < 0.01). This functional dependency appeared to be disturbed in dogs with PDH, as in these dogs CSF CRH concentrations did not correlate with plasma ACTH concentrations. It is concluded that continuous hyperstimulation of pituitary corticotropes with hypothalamic CRH is probably not the cause of excessive ACTH secretion in dogs with pituitary-dependent hyperadrenocorticism.


The segmental dorsal ramus neuropathy as a common cause of chronic and recurrent low back pain. The causes of recurrent and chronic low back pain usually remain unknown. The dorsal ramus lesion was found in 57 of 200 low back pain patients in this study in L5 or L4 segment without any neurophysiologic or neuroradiologic evidence of proximal ventral root compression. The neuropathy of dorsal rami, especially in their medial branches seems to be surprisingly common finding associated with low back pain and referred symptoms.


Development of gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and recovery are not coupled to inositol phosphate production or GnRH receptor number. After initial GnRH pretreatment (10 nM, 5 h), subsequent GnRH-stimulated LH release from the gonadotrope was diminished (1 microM GnRH stimulated release of 36.4 +/- 1.4% total cellular LH over 3 h in cells initially pretreated with medium alone compared to 27.4 +/- 1.2% in GnRH-pretreated cells); however, inositol phosphate (IP) production in response to the releasing hormone remained unaffected (1 microM GnRH provoked IP accumulation of 161 +/- 9% above basal levels after 45 min in control cells and 162 +/- 11% in GnRH-pretreated cells). Pretreatment of pituitary cell cultures with NaF (a guanyl nucleotide binding protein activator, 10 mM, 3 h) also decreased subsequent GnRH-stimulated LH release, and in addition, provoked a decrease in GnRH receptor number, an increase in GnRH receptor affinity, reduction of GnRH-stimulated IP production to basal levels, and an increase in the amount of LH released in response to stimulation with the calcium ionophore A23187. In order to determine if the changes in LH release were a result of decreased IP production and/or decreased GnRH receptor binding, the time course of recovery to control levels of these processes was assessed. GnRH receptor binding continued to decrease after NaF pretreatment, reaching a nadir (62% of control) at 6 h after the pretreatment period and recovering at 48 h (90% of control). In contrast, GnRH-provoked IP accumulation did not return to control levels even after 48 h of recovery after NaF pretreatment (1 microM GnRH-stimulated IP accumulation in NaF-pretreated cells was 57% compared to control cells after 48 h of recovery). GnRH-stimulated LH release was inhibited immediately after NaF pretreatment (1 microM GnRH-stimulated LH release in NaF-pretreated cells was 65% of control levels). Cells began to recover within 3 h (80% of control) and were almost completely recovered by 6 h (90% of control). A23187-provoked LH release was enhanced immediately after NaF pretreatment (30 microM A23187-stimulated LH release in NaF-pretreated cells was 170% of control levels). Responsiveness to ionophore was 133% of control by 0.5 h, and complete recovery was measured within 1 h (100% of control). Furthermore, both NaF and GnRH pretreatment still provoked a decrease in gonadotrope responsiveness when IP production was inhibited by the phospholipase C inhibitor U-73122. The results suggest that the development of gonadotrope desensitization (by either NaF or GnRH pretreatment) can be uncoupled from changes in IP production.(ABSTRACT TRUNCATED AT 400 WORDS)


Peripheral neuropathy in myotonic dystrophy: electrophysiological and clinical features. Peripheral neuropathy was investigated in thirty-one patients with myotonic dystrophy (MyD) and sixteen relatives. Using standard electrophysiological criteria, a sensorimotor axonal peripheral neuropathy was found in 14 MyD cases (45%) and not in unaffected first-degree relatives. The whole group of the MyD patients showed significant impairment of mean motor and sensory conduction values, compared with controls. The presence of polyneuropathy was correlated with the patients' age and the severity and duration of the clinical manifestations of MyD.


Expression of inhibin beta A and beta B, follistatin and activin-A receptor messenger ribonucleic acids in the rat seminiferous epithelium. The expression of inhibin beta A and beta B subunits, follistatin, and activin-A receptor messenger RNA (mRNAs) in different stages of rat seminiferous epithelial cycle was analyzed by in situ hybridization in order to understand their role in the regulation of spermatogenesis. Inhibin beta A mRNA was expressed in Sertoli cells in a highly stage-specific manner. The mRNA levels started to accumulate in Sertoli cells at stage VIII of the cycle and were highly expressed during stages IX-XI. Follistatin mRNA expression was identical to that of inhibin beta A, while inhibin beta B mRNA was maximally expressed in Sertoli cells at stages XIII-III. Low expression was found in stages VII-VIII. Activin-A receptor mRNA was localized mainly in spermatogenic cells. Maximal expression was seen in late primary spermatocytes at stages XIII-XIV and in early round spermatids at stages I-IV. A low even expression by Sertoli cells was also seen. Inhibin beta A and follistatin mRNAs were coexpressed in stage IX-XI Sertoli cells, suggesting close interplay between these molecules. The pattern of inhibin beta B mRNA expression was similar to that of inhibin alpha-mRNA. Localization of activin-A receptor mRNA in spermatogenic cells suggests that activin may influence meiotic divisions and early spermiogenesis.


Opposite regulation of deoxyribonucleic acid synthesis and iodide uptake in rat thyroid cells by basic fibroblast growth factor: correlation with opposite regulation of c-fos and thyrotropin receptor gene expression. Basic fibroblast growth factor (bFGF) increases DNA synthesis in rat FRTL-5 thyroid cells, as measured by increased incorporation of tritiated thymidine into DNA. We show that this action is associated with the ability of bFGF to increase cytosolic Ca2+ levels and transiently increase c-fos mRNA levels. Other agents that increase c-fos mRNA levels and DNA synthesis in FRTL-5 cells include TSH, insulin, insulin-like growth factor-I, phorbol esters, A23187, and alpha 1-adrenergic agents; the last two agents also act by increasing cytosolic Ca2+ levels. Despite its enhancement of DNA synthesis, however, bFGF decreases TSH-induced cAMP-mediated iodide uptake. This action appears to reflect two separate actions of bFGF. First, bFGF decreases TSH receptor mRNA levels and the ability of TSH to acutely increase cAMP levels in FRTL-5 cells. The ability of bFGF to negatively regulate TSH receptor mRNA levels is additive to and independent of the ability of TSH and its cAMP signal to negatively autoregulate TSH receptor mRNA levels. This is consistent with the effect of bFGF on cytosolic Ca2+ levels and the ability of increased cytosolic Ca2+ to decrease TSH receptor mRNA levels. Second, bFGF inhibits cAMP signal expression, as evidenced by its ability to inhibit (Bu)2cAMP-induced iodide uptake in FRTL-5 cells. Both effects are, presumably, associated with the ability of bFGF to counteract TSH/cAMP-induced increases in thyroid peroxidase mRNA levels, which we demonstrate. We suggest, therefore, that bFGF causes opposite effects on DNA synthesis and iodide uptake because of its effect on cytosolic Ca2+ levels and because increases in cytosolic Ca2+ can have opposite effects on gene transcription, particularly in the case of the TSH receptor and c-fos genes.


Lowered responsiveness of the catalyst of adenylyl cyclase to stimulation by GS in heterologous desensitization: a role for adenosine 3',5'-monophosphate-dependent phosphorylation. Treatment of chick hepatocytes with glucagon results in homologous and heterologous desensitization of the receptor-stimulated adenylyl cyclase. The loci of postreceptor heterologous desensitization was studied. The addition of excess purified GS to glucagon-desensitized hepatocyte membranes did not fully restore fluoride stimulation of adenylyl cyclase, even though the absolute activity was increased at least 2-fold. Treatment of chick hepatocytes with 8-bromo-cAMP resulted in a similar reduction of fluoride stimulation that could not be restored by the addition of purified GS. When membranes from control and glucagon-treated hepatocytes were treated with purified catalytic subunit of protein kinase-A (PKA), fluoride stimulation was lowered in control, but not glucagon-treated, membranes. Treatment of membranes from S49 kin- lymphoma cells with PKA also resulted in decreased fluoride- and forskolin-stimulated adenylyl cyclase activity, but activity stimulated by Mn2+ was not altered. Since previous studies from our laboratory had shown that GS and G(i) are not substrates for protein kinase-A, it appears that the catalyst of adenylyl cyclase is the likely locus of modulation. To determine if both chick hepatocytes and S49 cells contain similar types of adenylyl cyclase that could account for the similar PKA regulatory properties, we used polymerase chain reaction-based techniques to identify GS-stimulated adenylyl cyclases present in these systems. The chick liver contains both type 5 and type 6 adenylyl cyclases, while S49 cells contain the type 6 enzyme. Type 5 and 6 adenylyl cyclases are members of one widely expressed subfamily of mammalian GS-responsive adenylyl cyclases and share a predicted PKA phosphorylation site in the central cytoplasmic loop. This site is not found in other known adenylyl cyclases (types 1-4), although the olfactory-specific type 3 enzyme has a predicted site nearby. These data indicate that one component of hormone-induced desensitization of the adenylyl cyclase system can be at the level of the catalyst, where PKA-mediated phosphorylation could result in lowered responsiveness. The types 5 and 6 adenylyl cyclases are likely candidates for such regulation.


Growth hormone-releasing hormone is produced by rat Leydig cell in culture and acts as a positive regulator of Leydig cell function. Rat GH-releasing hormone (GHRH), mainly contained in hypothalamic neurons, has also been identified in several extraneural tissues, including the gastrointestinal tract, placenta, ovary, and testis. In the testis, GHRH mRNA is ontogenically regulated, and GHRH immunoreactivity can be observed in interstitial cells and tubules, suggesting an intratesticular role for the peptide. Leydig cells in culture are able to produce hypothalamic releasing hormones, i.e. CRH, which acts as an autocrine negative regulator of Leydig cell function. In this study we investigated whether GHRH is present in Leydig cells and evaluated the role of the peptide in Leydig cell function. Adult Leydig cells in culture produced considerable amounts of immunoreactive GHRH [23.9 +/- 2.1 (+/- SE) pg/10(6) cells.30 min], and the release of the peptide was acutely stimulated by hCG. HPLC analysis of GHRH in media from basal and hCG-treated cultures showed the presence of a single peak eluting at the same retention time as that of hypothalamic rat GHRH. Radioligand binding and activation studies revealed a common receptor for vasoactive intestinal peptide (VIP) and rat GHRH in Leydig cell membrane. Specific binding of [125I]VIP to Leydig cell membranes showed the presence of a single site, with high affinity and low binding capacity. The relative potencies of VIP-related peptides for inhibition of radioligand binding were: VIP > rat GHRH > secretin > human GHRH. In cultured Leydig cells, GHRH and VIP stimulated cAMP production, consistent with coupling of the receptor to the adenylate cyclase system. VIP displayed a lower ED50 than GHRH in stimulating cAMP production (P < 0.01), comparable with the higher binding potency of this peptide. No additive effects of VIP- and GHRH-stimulated cAMP generation were observed, suggesting that both peptides compete for the same receptor protein. GHRH and VIP had no effect on basal steroidogenesis, indicating a lack of tonic actions and compartmentalization of the peptides' effect. On the other hand, GHRH acted as a potentiator of the acute gonadotropin stimulation of testosterone production and cAMP generation. [125I]hCG binding to the Leydig cells in culture showed that GHRH was unable to affect the number or affinity of binding sites for hCG, indicating that the GHRH-sensitizing effect on LH action is beyond the level of gonadotropin binding and possibly is through the facilitation of LH receptor coupling functions.(ABSTRACT TRUNCATED AT 400 WORDS)


Pituitary corticotrope tumor (AtT20) cells as a model system for the study of early inhibition by glucocorticoids. The utility of the established ACTH secreting mouse pituitary tumor cell line AtT20 for investigating early glucocorticoid inhibition was examined. Three different strains of the cell line D1, D16v, and D16:16, respectively, were analyzed. In initial studies CRF and phorbol esters were used as secretagogues to examine the properties of hormone secretion. In a perifusion system (cells in suspension) D1 cells failed to respond to the secretagogues, whereas both D16v and D16:16 cells were responsive. However, hormone release declined upon repeated exposure to secretagogue in both D16v and D16:16 cells and similar data were obtained when cells adhering to cover slips were perifused. In static incubation D16:16 cells gave more consistent results especially with respect to inhibition by glucocorticoids and were used in all subsequent studies. Synthetic glucocorticoids acting through the type II receptor inhibited CRF-induced ACTH release within 45 min; at 120 min, stimulated release was strongly (80-90%) suppressed. In contrast, no consistent inhibition by corticosterone could be found. In the presence of glycyrrhetinic acid, an inhibitor of 11 beta-hydroxysteroid dehydrogenase, a high concentration of corticosterone (10 microM) did produce a slight inhibition of ACTH release. Dexamethasone also inhibited ACTH release induced by the calcium channel activator compound (+)202-791. The accumulation of cAMP in response to CRF was not altered by dexamethasone. The inhibitory effect of synthetic glucocorticoids on ACTH release was prevented by blockers of messenger RNA (actinomycin D, dichlorobenzimidazole ribofuranoside) or protein (puromycin) biosynthesis, indicating the induction of new proteins. Immunoblotting for lipocortin I (annexin I) and chromogranin A revealed no induction by dexamethasone of any of these proteins in D16:16 cells. Messenger RNA encoding lipocortin I was not detectable and was not induced by treatment with dexamethasone in D16:16 cells. These data show that the AtT20 D16:16 strain is a useful model for early glucocorticoid action, which is mediated by type II receptors and involves the induction of new protein(s). Notably, induction of lipocortin I messenger RNA or protein could not be detected at a time when the inhibitory effect of glucocorticoids on stimulated hormone secretion was maximal.


Mu- and kappa-opiate receptor control of prolactin secretion in rats: ontogeny and interaction with serotonin. The present study explores developmental changes in mu- and kappa-opiate receptor control of PRL secretion. The ontogeny of mu- and kappa-receptor function was determined by assessing the PRL response to the mu-agonist sufentanil (SUF) and the kappa-agonist U50488 in 5-, 10-, 15-, 20-, and 60-day-old rats. Both mu- and kappa-agonists stimulated PRL secretion at all ages. The selectivity of the mu- and kappa-agonists was confirmed by selective blockade with their respective antagonists (beta-funaltrexamine and nor-binaltorphimine). Serotonin mediation of opiate-induced changes in PRL secretion was explored across ontogeny by testing cyproheptadine (CYPRO) blockade of agonist responses in 5-, 10-, 20-, and 60-day-old rats. CYPRO attenuated the PRL response to the mu-agonist SUF in 20- and 60-day-old rats, but not in the 5- or 10-day-old pups. CYPRO did not block the kappa-agonist U50488 at any age. Similarly, pretreatment with parachlorophenylalaine lowered the PRL response to SUF in 60-day-old rats, but not in 10-day-old rats. These results support previous reports of a serotonin-mediated mu control of PRL secretion that develops by day 20 and a kappa control of PRL secretion that is independent of serotonin at all ages. These findings also suggest that a previously reported serotonin-independent mu-receptor-mediated control of PRL secretion can be stimulated early in ontogeny.


High density lipoproteins stimulate molecular weight 80K protein phosphorylation in human trophoblast cells: evidence for a protein kinase-C-dependent pathway in human placental lactogen release. Previous studies from our laboratory have demonstrated that high density lipoprotein (HDL) HDL and apolipoprotein-AI (apoAI) stimulate human placental lactogen (hPL) release from human trophoblast cells. To determine whether protein kinase-C (PKC) activation is involved in the mechanism of HDL- and apoAI-mediated hPL release, we examined the effects of these factors on the phosphorylation of cytosolic proteins known to be phosphorylated in response to PKC activation by phorbol myristate acetate (PMA). HDL and apoAI each caused a dose- and time-dependent increase in phosphorylation of a PMA-inducible 80K mol wt acidic cytosolic protein in a manner similar to that observed in many other cell types. Stimulation of 80K protein phosphorylation was apparent 5 min after the addition of HDL, apoAI, or PMA and was maximal at 15 min. Maximal 80K protein phosphorylation in cells exposed to PMA (1.6 microM), HDL (1500 micrograms/ml), and apoAI (600 micrograms/ml) was 284%, 206%, and 239% that in untreated cells, respectively. The increase in both 80K protein phosphorylation and hPL release in response to apoAI was prevented by pretreatment of the cells with the PKC inhibitor staurosporine (10 microM) or by down-regulation of PKC after extended preincubation of the cells with 16 microM PMA. (Bu)2cAMP and the adenylate cyclase activator forskolin, which stimulate hPL release, had no effect on 80K protein phosphorylation. These results strongly suggest that HDL- and apoAI-stimulated hPL release involves a PKC-dependent pathway. Since earlier studies also implicate a cAMP-mediated pathway in the stimulation of hPL release by these agents, it appears that multiple intracellular pathways are involved in the stimulation of hPL release.


A cholesteryl ester hydrolase inhibitor blocks cholesterol translocation into the mitochondria of MA-10 Leydig tumor cells. The present studies describe an unexpected action of a cholesteryl ester hydrolase inhibitor on MA-10 Leydig tumor cells. These studies were initially intended to use the inhibitor, diethylumbelliferyl phosphate, to block cholesteryl ester hydrolysis and, thus, determine the contributions of this form of cholesterol to steroidogenesis and reveal any direct hormone effects on cholesterol esterification. Although this compound acted as an effective inhibitor of the cholesteryl ester hydrolase in intact MA-10 cells, it inhibited steroidogenesis at lower concentrations and to a greater extent than could be explained by simple inhibition of the ester hydrolase enzyme. This compound proved not to be generally toxic, but blocked some process occurring between cAMP formation and cholesterol side-chain cleavage. The diethylumbelliferyl phosphate block of steroidogenesis was readily bypassed by 22-hydroxycholesterol. These data indicated that the compound inhibited cholesterol transport. The lesion in cholesterol transport was not general, but very specific; cholesterol translocation to the mitochondrial site of cholesterol side-chain cleavage was blocked by this organophosphate compound.


Polycyclic aromatic hydrocarbon metabolism in rat adrenal, ovary, and testis microsomes is catalyzed by the same novel cytochrome P450 (P450RAP). A novel ACTH-inducible P450, cytochrome P450RAP, is responsible for polycyclic aromatic hydrocarbon (PAH) metabolism in male rat adrenal microsomes. P450RAP is present at similar levels in male and female adrenal microsomes and is immunochemically distinct from P450IA1. Anti-P450RAP immunoblots a protein present in ovarian and testicular microsomes that is the same size as P450RAP and which coelutes with the P450 fraction during chromatography on an immobilized artificial membrane column made with phosphatidylcholine. Rat adrenal, ovarian, and testicular microsomes exhibit similar regioselectivities in the metabolism of two polycyclic aromatic hydrocarbons, dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BP). Unlike P450IA1, these microsomes form little or no 7-OH-DMBA and BP-4,5-diol but do catalyze the formation of a high proportion of the presumptive procarcinogen, DMBA-3,4-diol. The relative activities of PAH metabolism by untreated adrenal, testicular, and ovarian microsomes are approximately 60, 20, and 6 pmol/mg microsomal protein/min, respectively. PMSG induced PAH metabolism 2- to 5-fold in ovarian microsomes and also increased the P450RAP immunoblot. Hypophysectomy reduced PAH metabolism 3-fold in testicular microsomes while also decreasing the P450RAP immunoblot. This close correlation between PAH metabolism and expression of P450RAP indicates the involvement of the cytochrome in this activity. DMBA and BP metabolism by PMSG-treated rat ovarian microsomes and untreated testicular microsomes are each completely inhibited by anti-P450RAP but are not inhibited by anti-P450IA1. Essentially all of the PAH metabolism in rat adrenal, testis, and ovary is, therefore, catalyzed by P450RAP, which is hormonally elevated in each tissue by a variety of possible mechanisms, including induction and selective proliferation of cells that express this protein.


Beneficial effects of high daily dose bromocriptine treatment in Cushing's disease. Treatment with a high daily dose bromocriptine was evaluated in 6 Cushing's disease patients (4 females and 2 males; aged 23 to 56 years). The highest doses administered were 40 mg to patient 1, 55 mg to patient 2, 35 mg to patient 3, 25 mg to patient 4, 25 mg to patient 5, and 17.5 mg to patient 6. The former 3 cases, 2 (patients 1 and 2) of whom were previously reported and further followed up, showed clinical and biochemical improvement with the regimen. Patient 1 who obtained remission with 40 mg/day has been on remission for further 14 months with a total of 36 months. Patient 2, who had a reduction in pituitary tumor size with 35 mg daily, relapsed thereafter. The therapy, however, resolved the paradoxical responses of plasma ACTH and cortisol to arginine. Readministration of bromocriptine resulted into another clinical and biochemical improvement with 45 to 55 mg/day. Patient 3, a relapsed case after a remission with reserpine plus pituitary irradiation, showed an improvement in the 24-h urinary free cortisol excretion with 35 mg/day. Patient 4 was the only case who had a marked decrease in plasma cortisol (basal; 16.3, nadir; 1.9 micrograms/dl) after a single-dose bromocriptine test among the 5 cases tested. The patient had favorable response with 25 mg/day for 2 months but the dose was not increased after an escape. Patient 5 received the drug in 4 occasions, 7.5 to 25 mg/day, in combination with several agents, which failed to induce clinical remission. The last patient did not respond to a maximum dose of 17.5 mg/day. These observations suggest that, regardless of the result of a single-dose bromocriptine test, treatment with a high daily dose of bromocriptine, 35 mg or more, may be necessary to obtain a favorable clinical response and normal cortisol secretion.


Substrate-analogue-induced changes in the nickel-EPR spectrum of active methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum. Methyl-coenzyme-M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M [2-(methylthio)ethanesulfonate] and 7-mercaptoheptanoylthreonine phosphate in methanogenic archaea. The enzyme contains the nickel porphinoid coenzyme F430 as a prosthetic group. In the active, reduced (red) state, the enzyme displays two characteristic EPR signals, MCR-red1 and MCR-red2, probably derived from Ni(I). In the presence of the substrate methyl-coenzyme M, the rhombic MCR-red2 signal is quantitatively converted to the axial MCR-red1 signal. We report here on the effects of inhibitory substrate analogues on the EPR spectrum of the enzyme. 3-Bromopropanesulfonate (BrPrSO3), which is the most potent inhibitor of MCR known to date (apparent Ki = 0.05 microM), converted the EPR signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal designated MCR-BrPrSO3. 3-Fluoropropanesulfonate (apparent Ki < 50 microM) and 3-iodopropanesulfonate (apparent Ki < 1 microM) induced a signal identical to that induced by BrPrSO3 without affecting the line shape, despite the fact that the fluorine, bromine and iodine isotopes employed have nuclear spins of I = 1/2, I = 3/2 and I = 5/2, respectively. This finding suggests that MCR-BrPrSO3 is not the result of a close halogen-Ni(I) interaction. 7-Bromoheptanoylthreonine phosphate (BrHpoThrP) (apparent Ki = 5 microM), which is an inhibitory substrate analogue of 7-mercaptoheptanoylthreonine phosphate, converted the signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal, MCR-BrHpoThrP, similar but not identical to MCR-BrPrSO3. The results indicate that inhibition of MCR by the halogenated substrate analogues investigated above is not via oxidation of Ni(I)F430. The different MCR EPR signals are assigned to different enzyme/substrate and enzyme/inhibitor complexes.


EPR studies of cytochrome aa3 from Sulfolobus acidocaldarius. Evidence for a binuclear center in archaebacterial terminal oxidase. The purified cytochrome aa3-type oxidase from Sulfolobus acidocaldarius (DSM 639) consists of a single subunit, containing one low-spin and one high-spin A-type hemes and copper [Anemüller, S. and Schäfer, G. (1990) Eur. J. Biochem. 191, 297-305]. The enzyme metal centers were investigated by electron paramagnetic resonance spectroscopy (EPR), coupled to redox potentiometry. The low-spin heme EPR signal has the following g-values: gz = 3.02, gy = 2.23 and gx = 1.45 and the high-spin heme exhibits an almost axial spectrum (gy = 6.03 and gx = 5.97, E/D < 0.002). In the enzyme as isolated the low-spin resonance corresponds to 95 +/- 10% of the enzyme concentration, while the high-spin signal accounts for only 40 +/- 5%. However, taking into account the redox potential dependence of the high-spin heme signal, this value also rises to 95 +/- 10%. The high-spin heme signal of the Sulfolobus enzyme shows spectral characteristics distinct from those of the Paracoccus denitrificans one: it shows a smaller rhombicity (gy = 6.1 and gx = 5.9, E/D = 0.004 for the P. denitrificans enzyme) and it is easier to saturate, having a half saturation power of 148 mW compared to 360 mW for the P. denitrificans protein, both at 10 K. The EPR spectrum of an extensively dialyzed and active enzyme sample containing only one copper atom/enzyme molecule does not display CuA-like resonances, indicating that this enzyme contains only a CUB-type center. The EPR-redox titration of the high-spin heme signal, which is assigned to cytochrome a3, gives a bell shaped curve, which was simulated by a non-interactive two step redox process, with reduction potentials of 200 +/- 10 mV and 370 +/- 10 mV at pH = 7.4. The decrease of the signal amplitude at high redox potentials is proposed to be due to oxidation of a CUB(I) center, which in the CUB(II) state is tightly spin-coupled to the heme a3 center. The reduction potential of the low-spin resonance was determined using the same model as 305 +/- 10 mV at pH = 7.4 by EPR redox titration. Addition of azide to the enzyme affects only the high-spin heme signal, consistent with the assignment of this resonance to heme a3. The results are discussed in the context of the redox center composition of quinol and cytochrome c oxidases.


Expression, purification and characterisation of a functional phosphatidylinositol-specific phospholipase C-delta 1 protein in Escherichia coli. Inositol-lipid-specific phospholipase C-delta 1 (PtdIns-PLC delta 1) was expressed in Escherichia coli as a fusion protein containing a short 22-amino-acid lac-Z-derived amino terminus. Under appropriate conditions, the phospholipase constituted approximately 0.2% of the detergent-soluble protein and could be purified to near homogeneity in a simple three step protocol. The catalytic properties of the purified enzyme closely resemble those of the eukaryote-derived protein. The suitability of bacterial expression for the investigation of PtdIns-PLC delta regulation is discussed.


Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain. It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNA(Tyr) species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNA(Tyr) species were spliced. In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNA(Tyr) domains from yeast and Xenopus, respectively. The chimeric pre-tRNA comprising the mature tRNA(Tyr) domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNA(Tyr) domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.


1H-15N-NMR studies of bacteriorhodopsin Halobacterium halobium. Conformational dynamics of the four-helical bundle. Series of uniformly and selectively 15N-labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a 1H-15N-NMR study was performed in methanol/chloroform (1:1) and 0.1 M NH4CHOO, medium which mimics that in the membrane in vivo. Less than half of the cross-peaks expected from the amino acid sequence of uniformly 15N-labeled bacteriorhodopsin were observed, using heteronuclear 1H-15N coherence spectroscopy. In order to assign the observed cross-peaks, a selective 15N-labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and 1H-15N-NMR spectra of bacteriorhodopsin and its fragments C1 (residues (72-231), C2 (residues 1-71), B1 (residues 1-155) and BP2 (residues 163-231) were investigated. By this procedure, all observed 1H-15N cross-peaks of the entire bacteriorhodopsin were found to belong to the transmembrane segments A, B and G. The cross-peaks from four (C, D, E and F) helical bundles (79-189 residues) were missed. These results clearly indicate that dynamic processes occur in the four helice bundle. The significance of this, in respect to bacteriorhodopsin functioning, is discussed.


1H-NMR study of neurotoxin B-IV from the marine worm Cerebratulus lacteus. Solution properties, sequence-specific resonance assignments, secondary structure and global fold. Sequence-specific resonance assignments are reported for the 500-MHz 1H-NMR spectrum of the 55-residue neurotoxin B-IV, isolated from the heteronemertine worm Cerebratulus lacteus. A range of two-dimensional homonuclear correlated and NOE spectra was used in making these assignments, which include NH, C alpha H and C beta H resonances, as well as most resonances from longer-chain spin systems, with the exception of the ten Lys residues, where spectral overlap prevented complete, unambiguous assignments. The secondary structure of B-IV was identified from the pattern of sequential (i, i + 1) and medium range (i, i + 2/3/4) NOE connectivities and the location of slowly exchanging backbone amide protons. Two helices are present, incorporating residues 13-26 and 33-49, and the C-terminal five residues form a helix-like structure. A type-I reverse turn, involving residues 28-31 is present in a small loop linking the two major helices, and the N-terminus appears to be unordered at 27 degrees C, although it may adopt a more ordered conformation at lower temperatures. These elements of secondary structure, together with the four disulfide bonds in the protein, provide sufficient information to define the global fold of the molecule in solution. The pH and temperature dependence of the toxin have been investigated by 1H-NMR and the pKa values of several ionisable sidechains determined.


Voltage dependence of liver gap-junction channels reconstituted into liposomes and incorporated into planar bilayers. The voltage dependence of rat liver gap junctions was investigated using non-denaturing solubilization and reconstitution of gap-junction protein into proteoliposomes in controlled conditions of connexon aggregation. The presence of liver connexin 32 in reconstituted proteoliposomes was checked with specific antibodies. The proteoliposomes were inserted into planar lipid bilayers by fusion. The single-channel conductance was voltage independent, and its magnitude was 700-1900 pS in 1 M NaCl, as expected from other reports, assuming that conductance is linear with ion activity. The channels were open at zero voltage and completely closed above 40 mV in either direction. This steep voltage dependence corresponded to an open/closed-state voltage difference of 19 mV and to 3.5 gating charges moving through the field. When several channels were inserted into the bilayer, a large fraction of the membrane conductance became voltage insensitive. These results show that the isolated channel units are highly voltage dependent and are consistent with the assumption that aggregated connexons interact through links which prevent voltage-sensitive conformational changes.


Beta-agonist-induced inhibitory-guanine-nucleotide-binding regulatory protein coupling to adenylate cyclase in mollusc Anodonta cygnea foot muscle sarcolemma. In the sarcolemma fraction of foot muscles of a fresh-water bivalve mollusc, Anodonta cygnea, a direct inhibitory, rather than stimulatory, effect of the beta-adrenergic agonist isoproterenol, at micromolar concentration, on cAMP level and adenylate cyclase activity, was revealed. It was blocked by beta- but not alpha-adrenergic antagonists. A single class of [3H]dihydroalprenolol-binding sites with binding properties of beta-adrenergic receptor was detected in mollusc sarcolemma. Potentiation of the inhibitory effect of isoproterenol on mollusc adenylate cyclase activity by GTP or guanosine 5'-[beta,gamma-imido]triphosphate at micromolar concentrations, and its elimination in the presence of guanosine 5'-[beta-thio]diphosphate, were shown. The pertussis-toxin-induced ADP-ribosylation of sarcolemma 40-kDa protein [immunochemically related in the C-terminal part to pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein (G-protein) alpha subunits of vertebrates], as well as the treatment of mollusc sarcolemma with antisera responsive to the C-terminus of vertebrate inhibitory G-protein (G(i)) alpha subunit led to elimination of the inhibitory effect of isoproterenol on adenylate cyclase activity. The results obtained suggest that beta-agonist-induced inhibition of adenylate cyclase in A. cygnea foot muscle may be realized via the beta-adrenoreceptor/G(i) signalling pathway.


Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation. Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gamma S]), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gamma S] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP[gamma S] and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)


1H-NMR study on the chitotrisaccharide binding to hen egg white lysozyme. Interaction between hen egg white lysozyme and chitotrisaccharide was investigated by 1H-NMR spectroscopy using partially acetylated chitotrisaccharides and chemically modified lysozyme. Monoacetyl (GlcN-GlcN-GlcNAc), diacetyl (GlcN-GlcNAc-GlcNAc), or triacetyl chitotrisaccharide [(GlcNAc)3] was added to the lysozyme solution, and the changes in the 1H-NMR signals of the lysozyme were analyzed. Although many of the resonances were affected by addition of the saccharide, the most remarkable effect was seen on the signal of Trp28 C5H which is in a hydrophobic box adjacent to the saccharide-binding site. The signal shifted upfield by 0.2 ppm upon (GlcNAc)3 binding, whereas the chemical shift change of the signal resulting from binding of GlcN-GlcNAc-GlcNAc or GlcN-GlcN-GlcNAc was smaller than that resulting from (GlcNAc)3 binding. When the Asp101-modified lysozyme was used instead of the native lysozyme, the chemical shift change of the Trp28 C5H signal resulting from (GlcNAc)3 binding was also smaller than that for the native lysozyme. The chemical shift change of the signal reflects the conformational change of the hydrophobic box region which should synchronize with the movement of the binding site resulting from the saccharide binding. Therefore, the conformational change resulting from the saccharide binding might be reduced when the sugar residues located at binding subsites A and B of the lysozyme are deacetylated, as well as when Asp101 interacting with the sugar residues at the same subsites is modified.


Comparison of biochemical properties of DNA-topoisomerase I from normal and regenerating liver. Biochemical properties of topoisomerase I from normal and regenerating rat liver were analysed using crude or fractionated nuclear extracts. We could not detect significative change in topoisomerase I content or activity (magnesium stimulation and inhibition by ATP) during the course of liver regeneration. Topoisomerase I can be resolved into two species of 97 kDa and 100 kDa, with the same pI of 8.2-8.6 as shown by two dimensional gel electrophoresis. The two polypeptides contained a non-phosphorylated precursor and others forms with variable degrees of phosphorylation. In-vitro dephosphorylation with alkaline phosphatase leads to the disappearance of the phosphorylated forms and inactivation of the enzyme. The affinity of topoisomerase I for chromatin (measured by salt elution) differs markedly between normal and regenerating liver: nearly 50% of topoisomerase I remained bound to the chromatin from normal liver at 250 mM NaCl whereas it was completely eluted from 24-h-regenerating-liver nuclei. The biological significance of these results is discussed.


Developmental changes in hepatocyte growth factor mRNA and its receptor in rat liver, kidney and lung. Hepatocyte growth factor (HGF) is a mesenchymal-derived factor which induces mitosis, cell movement and morphogenesis of tissue-like structure. We analyzed changes in HGF mRNA and its receptor, the c-met proto-oncogene product, in the liver, kidney and lung during late fetal and postnatal development in rats. In the liver, the HGF-mRNA level was very low during late gestation and in neonates, it increased remarkably and reached a maximum two weeks postnatally, to be followed by a decrease to 33% of the maximum. HGF mRNA in the kidney and lung was either undetectable or very low during late gestation and the neonatal period and increased markedly to reach a maximum, respectively, 3-4 weeks postnatally. HGF-mRNA level in the adult rat lung was fivefold higher than that in the liver and kidney. The number of HGF receptors on plasma membranes of these tissues was low in neonates but there was a rapid increase after birth and a maximum was reached within three weeks. The number of HGF receptors/ng plasma membrane protein at the maximal level was highest in the liver and lowest in the lung. c-met/HGF-receptor mRNA in the liver was also low during late-gestation or in early neonatal periods and increased postnatally. Since HGF-mRNA and HGF-receptor levels changed differently in liver, kidney and lung, the expression of HGF and its receptor may be independently regulated in each organ. However, in these organs, HGF mRNA and the HGF receptor increased within a few weeks of birth, HGF may play roles in organ growth, organ maturation and the maintenance of tissue homeostasis during the postnatal period, presumably through its potential to act as mitogen, motogen and morphogen.


Binding of a 100-kDa ubiquitous factor to the human prolactin promoter is required for its basal and hormone-regulated activity. cAMP strongly stimulates the activity of the human prolactin (hPRL) promoter. We have previously shown that two types of cis-element are required for this cAMP regulation; binding sites for the pituitary-specific factor Pit-1, and the sequence spanning nucleotides -115 to -85 (named sequence A). Sequence A contains the TGACG motif found in the consensus sequence of the cAMP-responsive element (CRE). In this study, we show that a mutation in the TGACG motif of sequence A strongly reduces not only the cAMP regulation but also the Ca2+ regulation and basal activity of the hPRL promoter. Furthermore, gel-shift assays indicate that the mutation prevents binding of a ubiquitous factor which is not the CRE-binding protein. Southwestern experiments suggest that this ubiquitous factor's molecular mass is approximately 100 kDa. We conclude that binding of a 100-kDa ubiquitous factor to sequence A is required for full basal and hormonal regulation of hPRL-promoter activity.


Biological methane activation involves the intermediacy of carbon-centered radicals. The spin-trapping technique has demonstrated that carbon-centered radicals are produced during soluble-methane-monooxygenase catalysis of the hydroxylation of several different types of substrate. The resulting spin-adducts were identified from the hyperfine splitting constants in their EPR spectra. Isotopic labelling showed unequivocally that the trapped radicals were derived from substrate. The carbon-centered substrate radicals are believed to result from hydrogen-atom abstraction by a ferryl species in a cytochrome-P-450-like mechanism. No hydroxy radical nor an oxygen-based radical of any kind was detected in any of the spin-trapping experiments.


T lymphocytes mediate immunologic control of C3 gene expression. Immunologic control of C3 gene expression by tissue macrophages can be accomplished by treatment of spleen fragments with anti-C3 antibody. We now demonstrate that suppression of C3 requires participation of T lymphocytes of both the CD4+ and CD8+ phenotypes. Pretreatment of splenic tissue with anti-Thy-1.2 monoclonal antibody blocks the ability of the anti-C3 antibody to induce C3 suppression. Reduction in either the CD4+ or CD8+ subpopulations of T lymphocytes also abrogates C3 suppression demonstrating that both T cell subsets are required in addition to the inducing antibody. Artificially elevating intracellular levels of cAMP with cholera toxin can partially substitute for the effects mediated by T cells in this reaction. Therefore, normal expression of the C3 gene can be suppressed by a regulatory network that requires the presence of a specific inducing antibody and T lymphocytes of both the CD4+ and CD8+ subsets. This regulatory network has many similarities to regulatory networks that have been well documented in suppression of specific murine immunoglobulin allotypes.


Purification of the cardiac 1,4-dihydropyridine receptor using immunoaffinity chromatography with a monoclonal antibody against the alpha 2 delta subunit of the skeletal muscle dihydropyridine receptor. The 1,4-dihydropyridine receptor associated with L-type Ca2+ channels was purified about 1700-fold from porcine cardiac sarcolemmal membranes using a simple and rapid (ca. 8 h) two-step procedure: wheat germ agglutinin affinity chromatography followed by immunoaffinity chromatography with a monoclonal antibody (MCC-1) against the alpha 2 delta subunit of the skeletal muscle Ca2+ channel with a glycine elution buffer (pH 3). Gel electrophoresis of this purified sample under non-reducing conditions revealed a major polypeptide band with molecular weight of 190 kDa, which was separated under reducing conditions to a 155 kDa band and 2-3 bands with M(r) about 20 kDa, corresponding to alpha 2 and delta subunits, respectively. The peptide band corresponding to the alpha 1 subunit was not detected in this gel electrophoresis. However, the alpha 1 subunit without bound alpha 2 delta was selectively eluted from MCC-1 Sepharose with 1% Triton X-100. A 190 kDa band corresponding to the alpha 1 subunit was visualized by fluorography and by silver staining in the fraction eluted with Triton X-100. Electrophoretically, the amount of alpha 1 was smaller than that of the alpha 2 subunit in the purified sample obtained here.


Ca2+ modulates the inhibition of (+)-[3H]isradipine binding by amiloride and quinacrine. Binding studies were performed to characterize the inhibition by amiloride, 3,4-dichlorobenzamil and quinacrine of specific binding of (+)-[3H]isradipine to L-type voltage-operated calcium ion channels in rat cardiac membranes at 37 degrees C with and without 10(-3) M calcium added. By analysis of saturation, inhibition and dissociation curves we find that without the addition of calcium, amiloride (constant of inhibitor producing 50% inhibition (K0.5) = 6.9 x 10(-4) M, Hill coefficient (nH) = 1.99, k-1 increased) and 3,4-dichlorobenzamil (K0.5 = 7.7 x 10(-7) M, nH = 1.13, k-1 increased) inhibit (+)-[3H]isradipine binding by complex, allosteric interactions, suggesting positive cooperativity between sites for the inhibitors. Quinacrine (K0.5 = 6.7 x 10(-6) M, nH = 0.84, k-1 increased) inhibits the binding allosterically by an action compatible with binding to one site. Addition of 10(-3) M calcium affected the inhibition by amiloride (K0.5 = 1.02 x 10(-3) M, nH = 1.41) and quinacrine (K0.5 = 3.3 x 10(-5) M, nH = 0.65). With calcium added the mechanisms of inhibitions were complex, allosteric, and could be explained by positive cooperativity between sites for amiloride and negative cooperativity between sites for guinacrine. We conclude that calcium addition modulates the inhibitions by amiloride and quinacrine by increasing the inhibition constants and changing the cooperativity.


Opioid-controlled adenylate cyclase in the guinea-pig myenteric plexus is confined to nerve somata. Previous studies with the electrically stimulated longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum suggested that opioid control of adenylate cyclase is confined to nerve somata. No indication was found for an opioid effect on the enzyme at nerve terminals of the neuro-muscular junction. The aim of the present investigation was to directly study the effect of opioids on cAMP generation in nerve fragments associated with somata or terminals of the myenteric plexus. Employing the ultracentrifugation technique an enrichment of cell organelles in fractions relating to either somata or terminals was achieved. Opioid binding studies revealed specific mu-receptors which in both fractions were regulated by GTP. Challenge of these fractions with forskolin and prostaglandin E1, respectively, resulted in an increased production of cAMP regardless of their neuroanatomical assignment. Examining the response of neuronal material to the selective mu-opioid DAMGO ([D-Ala2, MePhe4, Gly-ol5]enkephalin) revealed an inhibitory action on cAMP synthesis in somata-enriched fractions. No effect of DAMGO was observed in material linked to nerve terminals, although the presence of mu-opioid receptors and adenylate cyclase has been demonstrated. We conclude that opioid control of adenylate cyclase in the myenteric plexus of the guinea pig is confined to nerve somata.


2,5-Di-(tert-butyl)-1,4-benzohydroquinone and cyclopiazonic acid decrease the Ca2+ permeability of endoplasmic reticulum. Specific inhibitors of the endoplasmic-reticulum Ca2+ pump will deplete intracellular stores and are therefore useful to study the role of store depletion on plasma-membrane Ca2+ permeability. We now report that the Ca(2+)-pump inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) reduces the passive Ca2+ leak from the internal stores in permeabilized A7r5 vascular smooth-muscle cells. This aspecific effect occurred at concentrations that are normally used to empty the stores in intact cells. Cyclopiazonic acid exerted a similar, although less pronounced effect, while thapsigargin did not affect the passive Ca2+ leak. The inositol 1,4,5-trisphosphate-mediated Ca2+ release was not affected. tBuBHQ and cyclopiazonic acid cannot therefore be used as specific tools to probe the mechanism of receptor-mediated Ca2+ entry.


Inhibition of active oxygen generation by dipyridamole in human polymorphonuclear leukocytes. The effect of dipyridamole on active oxygen generation by human polymorphonuclear leukocytes (PMN) was investigated. Dipyridamole inhibited the production of oxidative metabolites from human PMN stimulated by opsonized zymosan and formyl-methionyl-leucyl-phenylalanine dose and time dependently. To determine whether dipyridamole directly inhibits the production of oxygen metabolites by human PMN, human PMN were preincubated with dipyridamole washed prior to stimulation. Dipyridamole was found to directly inhibit human PMN from generated active oxygen metabolites at therapeutic concentrations. Dipyridamole may possibly be a potential scavenger of active oxygen metabolites since it inhibited active oxygen metabolite production from human PMN very rapidly. Dipyridamole was also found to directly affect the scavenging of active oxygen metabolites generated by opsonized zymosan-stimulated human PMN at therapeutic concentrations. This action of dipyridamole was also noted to be exerted against hydroxyl radicals and superoxide anions produced biochemically by an electron spin resonance spectrometer. It thus follows that dipyridamole may inhibit human PMN active oxygen metabolite generation and affect directly the scavenging of active oxygen metabolites at therapeutic concentrations.


Effects of endothelins on the human megakaryoblastic cell line MEG-01. Some effects of endothelin-1 (ET-1) were studied on the megakaryoblastic cell line MEG-01. ET-1 induced an elevation of the intracellular levels of Ca2+([Ca2+]i) as measured with the fluorescent indicator indo-1, which consists of an initial transient increase and an ensuing sustained plateau. The plateau phase was abolished by removal of extracellular Ca2+. In addition, ET-1 induced a rapid (within 5 s) increase in the accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and more delayed increases in Ins(1,3,4)P3 and Ins(1,3,4,5)P4, but did not modify cAMP levels. ET-1 homologues (ET-2, ET-3, sarafotoxin 6b and vasointestinal constrictor) also induced biphasic effects on [Ca2+]i. The Ca2+ elevation was concentration dependent, the order of potency being sarafotoxin 6b > ET-1 > ET-2 = vasointestinal constrictor > ET-3. The actions of ET analogs in raising [Ca2+]i were mutually exclusive, suggesting that they act through the same mechanism. These results suggest that cells of the megakaryoblast/megakaryocyte lineage are targets for endothelins.


The products of the reaction between toluene diisocyanate and water contract isolated guinea pig bronchi. We have investigated the ability of the products of the reaction between toluene diisocyanate (TDI) and water to contract bronchial smooth muscle. The experiments were performed in isolated guinea pig bronchi. TDI, both 2,4- and 2,6-toluenediamine (TDA) and mixtures of 2,4- and 2,6-TDA (ratio 80:20 and 20:80) caused concentration-dependent contraction in the isolated bronchi. The mixture of disubstituted urea and biuret also contracted the bronchi, but not in a concentration-dependent fashion. Our results provide evidence that all products of the reaction between toluene diisocyanate and water have the ability to contract isolated bronchial smooth muscle in guinea pigs. Whatever the role of toluenediamine in the adverse respiratory effects induced by exposure to isocyanates, our findings reveal the necessity of in vivo studies on the metabolism of inhaled toluene diisocyanate in humans to improve our understanding of the mechanism of action of isocyanates.


Autoradiographic study of peripheral benzodiazepine receptors in animal brain tumor models and human gliomas. In vitro binding of [3H]PK-11195 (1-(2-chlorophenyl)-N-methyl-(1- methylpropyl)-3-isoquinoline carboxamide) in rodent AA ascites and C6 glioma as well as in human gliomas was investigated. The Bmax (mean +/- S.D.) of AA ascites tumor and C6 glioma is 1.39 +/- 0.15 pmol/mg tissue and 4.50 +/- 0.76 pmol/mg tissue, respectively. This Bmax is 9 and 30 times, respectively, higher than the one found in the rat cortex (0.15 +/- 0.03 pmol/mg tissue). A Bmax of 1.26 +/- 0.24 pmol/mg tissue and 0.64 +/- 0.08 pmol/mg tissue was found in human malignant and low grade gliomas respectively. This Bmax value should be compared to 0.35 +/- 0.04 pmol/mg tissue found in the normal human cortex. There are significant (P less than 0.05) differences between Bmax in tumors and normal cortex. There was no significant difference in KD between the malignant and low grade gliomas. C6 glioma has a KD significantly greater than rat cortex. In some cases of human low grade gliomas, kinetic measurements suggested the presence of two affinity receptor sites. However, at this time, heterogeneity of the tissue cannot be excluded as being at least in part a source of this.


Multiple mechanisms for doxorubicin cytotoxicity on glomerular epithelial cells 'in vitro'. This study was planned to define the metabolic pathways for free radical production by isolated glomeruli and glomerular epithelial cells in vitro after exposure to cytotoxic doses of doxorubicin. A net increment in glomerular superoxide anion (O2.) synthesis was observed at doxorubicin doses between 10 and 30 micrograms/ml, a drug level which also induced a parallel increment in uric acid synthesis. Since the synthesis of O2. with production of uric acid implies an activity of xanthine oxidase, a few experiments were performed with glomeruli which had been deprived of xanthine oxidase activity. In this case doxorubicin-inducible O2. and uric acid synthesis by glomeruli was practically nil. A similar stimulatory effect of O2. synthesis was induced by doxorubicin on glomerular epithelial cells and also in this case O2. synthesis was suppressed by pre-treating cells with deoxyconformicin, a selective inhibitor of adenosine deaminase. Finally, equimolar amounts of the drug were equally cytotoxic even when kept constantly outside the cell by a stable linkage with an agarose macroporous bed. In summary, these data demonstrate that O2. is generated by isolated glomeruli and glomerular epithelial cells 'in vitro' when exposed to cytotoxic amounts of doxorubicin and that purine degradation to uric acid furnish the metabolic pathways for glomerular O2. generation. However, doxorubicin is comparably cytotoxic on glomerular epithelial cells from outside cells thus suggesting that also a membrane perturbation may activate the series of events leading to cell injury.


Species-specific binding of transformed Ah receptor to a dioxin responsive transcriptional enhancer. The Ah receptor (AhR) mediates many, if not all, of the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons. Although wide variations in species sensitivity to these compounds have been observed, numerous biochemical and physiochemical characteristics of the AhR appear similar among species. We have examined the ability of cytosolic AhR, from a variety of species (rat, rabbit, guinea pig, hamster, mouse, cow, sheep, fish, chicken and human), to transform and bind to its cognate DNA recognition sequence, the dioxin responsive enhancer (DRE), to evaluate the importance of these events in species variations in TCDD responsiveness. Gel retardation analysis using a murine DRE oligonucleotide has revealed that cytosolic AhR from a wide variety of species can transform in vitro and bind to the DRE and demonstrates that all of the factors necessary for AhR transformation and DNA binding are present in cytosol. In addition, DNA-binding analysis using a series of mutant DRE oligonucleotides has indicated no apparent species- or ligand-dependent, nucleotide-specific difference in AhR binding to the DRE. These studies support a highly conserved nature of the DRE and AhR (at least in DNA binding) and imply that a sequence closely related to the murine consensus DRE sequence is responsible for conferring AhR-dependent, TCDD responsiveness in each of these species.


Deletion of subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex specifically impairs electron transfer at the ubiquinol oxidase site (center P) in the bc1 complex. Deletion of QCR9, the nuclear gene encoding subunit 9 of the mitochondrial cytochrome bc1 complex in Saccharomyces cerevisiae, results in inactivation of the bc1 complex and inability of the yeast to grow on non-fermentable carbon sources. The loss of bc1 complex activity is due to loss of electron transfer activity at the ubiquinol oxidase site (center P) in the complex. Electron transfer at the ubiquinone reductase site (center N), is unaffected by the loss of subunit 9, but the extent of cytochrome b reduction is diminished. This is the first instance in which a supernumerary polypeptide, lacking a redox prosthetic group, has been shown to be required for an electron transfer reaction within the cytochrome bc1 complex.


Evidence for an interaction between cytosolic aldolase and the ATP-and pyrophosphate-dependent phosphofructokinases in carrot storage roots. Immunoaffinity chromatography was employed to identify potential plant cytosolic aldolase (ALDc) binding proteins. A clarified homogenate of carrot storage root was chromatographed on a column of protein-A-Sepharose that had been covalently coupled to anti-(carrot root ALDc) immunoglobulin G. The column was washed with phosphate-buffered saline (PBS), followed by step-wise elution with increasing concentrations of NaCl in PBS. Several proteins were eluted following application of the salt gradient. Western blotting identified the major eluting proteins to be the PPi-dependent phosphofructokinase (PFP) and the cytosolic form of the ATP-dependent phosphofructokinase (PFKc), enzymes that are metabolically sequential to ALDc. The results suggest that ALDc may specifically interact with PFP and PFKc in carrots.


ATP-activated Ca(2+)-permeable channels in rat peritoneal macrophages. The patch-clamp technique was used to study mechanisms of ATP-induced Ca2+ influx in rat peritoneal macrophages. The experiments on whole-cell and patch membranes have shown that extracellular ATP activates channels permeable to di- and monovalent inorganic cations. Ratios of unitary channel conductances in 105 mM Ca2+, Sr2+, Mn2+, Ba2+ and normal sodium solutions were 1.0, 0.95, 0.75, 0.55 and 0.85, respectively. The channels could open in the presence of non-hydrolyzable GTP analogues in artificial intracellular solution. The data are consistent with the hypothesis that a GTP-binding protein is involved in receptor-to-channel coupling.


ATP binding to bovine serum albumin.

ATP binding to bovine serum albumin. Specific binding of ATP to bovine serum albumin (BSA) is demonstrated employing ATP derivatives spin-labeled at either N6 or C8 of adenine ring or at the ribose moiety. Based on a 1:1 stoichiometry binding constants are in the 50-100 microM range. Binding is largely competitive with ATP or stearic acid. A small fraction of the labeled nucleotides could not be liberated by these ligands. Binding of AMP is in the millimolar range, only.


Internucleosomal chromatin degradation in myeloma and B-hybridoma cell cultures. The activity of Ca/Mg-dependent endonuclease (CME) is strongly inhibited in myeloma X-63.Ag8.653 and B-hybridoma MLC-1c as compared with mouse splenocytes. Nevertheless, pronounced internucleosomal chromatin degradation occurs in both cell lines during long-term cultivation without passing. In isolated cell nuclei of X-63 the activation of CME, which precedes chromatin fragmentation in vivo and loss of cell viability, is revealed. The time-course of CME activation is opposite to cell proliferation and is not accompanied by alterations in enzyme quantity. The results suggest that cell death of X-63 and MLC-1c occurs via apoptosis, and involves the mechanisms controlling the activation and/or interaction of CME with chromatin.


Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser86 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Ser86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.


[A quantitative analysis of primate motor activity in a situation of delayed spatial choice. The individual behavioral reactions]. Speed of movements in food getting responses of baboons, total time of the response, trajectories of the movements and other parameters revealed obvious changes during formation of conditioned activity. The data obtained suggest that many quantitative characteristics of the primate motor activity are specifically related to various aspects of complex behavioral responses.


[The contractile reaction of the myoepithelial cells in the mammary alveoli of the mouse to the multiple use of oxytocin]. The contractile response of the white mice mammary gland to oxytocin was found not to change with 5-min intervals of administration, whereas shortening of the interval to 1-2 min diminished the response. The myoepithelium seems to be able to contract with a higher frequency than during the milk ejection reflex in mice under natural conditions.


[The involvement of intracellular regulatory systems in the mechanisms of the recovery of the neuronal activity of the cerebral cortex in anoxia]. Changes in the membrane-bound calcium (Ca(b)), polyphosphoinositides (PPI) and cAMP, as well as unit spike activity of the cat brain cortex were studied during 30 min of recovery after 5-min anoxia. The cats with poor recovery revealed low Ca(b) and PPI but high level of the cAMP content. The group of cats with more successful restitution after anoxia revealed a sharp increase in the Ca(b) and PPI and not in the cAMP.


[Physical work capacity as a criterion of human resistance under the hot climatic conditions of an arid zone]. In heat discomfort, physically trained subjects had a high and stable physical working ability (PWA) whereas untrained subjects revealed a considerable decline of the parameter. A lesser work was performed with a greater tension of the cardiorespiratory system. The data obtained suggest a high and stable PWA is one of the main criteria of the organism's adaptation to hot climate conditions of the arid zone.


[The mechanism of the formation of the spatial-frequency selectivity of the neurons in area 21 of the cat cerebral cortex]. Spatial-frequency characteristics of receptive field (RF) neurons were studied in the cat area 21. The characteristics differed in different parts of the RFs. The data obtained reveal that the spatial-frequency tuning of RFs as a whole is the result of spatial interaction (algebraic summation) of different tunings of RF's different parts.


[GABA-immunoreactive structures in the lumbar spinal cord of the frog]. The distribution of GABA-immunoreactive (GABA-I) structures has been studied in the frontal sections of the lumbar spinal cord of the lake frog using PAP-method. GABA-I was revealed as labeled cell bodies, fragments of fibres and small point-like structures assumed to be in most cases synaptic contacts of GABA-I axons. Labeled fragments of fibres and point-like structures were found in gray and white matter of the spinal cord. The GABA-I cell bodies were revealed in the dorsal horn, intermediate zone and ventral part of the gray matter. Labeled cells were found in the cavity of the central canal. The findings are discussed in relation to the modern data on the GABA-ergic control of the spinal cord functions.


[The correction of a disorder in visual recognition in monkeys with the use of an antioxidant]. A normal differentiation between black and white visual stimuli lesioned by phthoracisine can be restored by oxymetacyl administration (3 mg/kg) in monkeys. The colour differentiation is restored after the dose 4 mg/kg. The data obtained suggest correcting effect of oxymetacyl.


[Activation of the heart rhythm during negative compression of the eyeballs in healthy subjects]. A phenomenon of oculosympathetic reflex (OSR) has been discovered: the activation of heart rate during an eyeball suction. The OSR seems to be due to either activation of eyeball proprioceptors mediated by the RF and locus coeruleus, or to a baroreflex mechanism originated from the intraocular portion of art. ophthalmica. The OSR and Aschner's reflex seem to trigger some vegetative barometeopathetic responses.


[The regulation of the lumen of the major arteries in accordance with the shear stress on the endothelium]. A decrease in the blood viscosity due to hemoconcentration, caused arterial dilatation. These viscosity-induced responses depended on intact endothelium. The dilatory responses caused by equal increments in flow rate, increased in systemic hemoconcentration and decreased in systemic hemodilution. The data obtained corroborate the suggestion that stress applied to endothelium is the key signal in the control of arterial lumen by the blood flow rate.


[Cholinergic suppression of the angiotensin I-converting reaction]. I. v. administration of carbachol or neostigmine produced a dose-dependent decrease in the angiotensin I pressor response and in conversion of angiotensin I to angiotensin II in anesthetized rats. Physostigmine produced the same effects in combination with propranolol. Administration of cholinomimetics with atropine leads to no such effect. The activation of the cholinergic system seems to inhibit a formation of angiotensin II.


[The microcirculatory changes in leukocytosis]. A moderate leucocytosis was found to decelerate the blood flow in the rat brain capillaries and to change the character of the blood flow. This seems to be due to a temporal obturation of the capillary lumen by one or several leucocytes.


[The reaction of a pregnant female and of her just-born rat pups to a change in ambient temperature and in the blood glucose level]. Cold exercises and hyperglycemia were found to exert a benevolent effect on the reproductive function and postnatal development of rats. Warmth and hyperglycemia during early periods of pregnancy led to a disturbance in the reproductive function and postnatal development in rats.


[Water-salt metabolism in the maternal and fetal organisms during hydration at different periods of the pregnancy]. The water surplus in the organism of a pregnant rat was found to cause an increase in the diuretic and ionouretic responses. The hyperhydration, particularly in the beginning of the pregnancy, was found to cause a high percentage of the foetus reabsorption.


Cell surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenal cortex. Previous studies demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) regulates steroid biosynthesis. In this study we investigate further PBR action by examining its subcellular localization in mouse adrenal gland using anti-peptide PBR antiserum and employing biotin-streptavidin peroxidase immunocytochemistry. Results demonstrated PBR immunostaining exclusively in the cortex. Within this region, however, PBR staining was homogeneously distributed in cells of the zona glomerulosa, whereas in cells of the zona fasciculata both cytoplasmic and prominent plasma membrane immunostaining was evident. Next, PBR distribution was examined using confocal microscopy. Confocal optical sections were obtained, 3-D reconstructions of these sections generated, and vertical, z-sections of the 3-D reconstruction recreated. The immunostaining pattern observed was consistent with a cell surface distribution of PBR. The demonstration of a subset of PBR at the plasma membrane may account for actions of PBR ligands not related to mitochondrial function.


Inhibition of insulin-like growth factor-I responses in MCF-7 cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Insulin-like growth factor-I (IGF-I) stimulated the growth and [3H]thymidine uptake in MCF-7 human breast cancer cells grown in serum- and growth factor-inactivated serum-containing media. Cotreatment of the cells with IGF-I plus 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a significant decrease in mitogen-induced cell proliferation and [3H]thymidine uptake. Similar effects were observed for cells treated with 2,3,7,8-TCDD and IGF-I plus 17 beta-estradiol. The relative antimitogenic activities of 2,3,7,8-TCDD and related compounds followed the order 2,3,7,8-TCDD greater than 2,3,7,8-tetrachlorodibenzofuran (TCDF) greater than 1,2,7,8-TCDF greater than 1,3,7,8-TCDD which was similar to their aryl hydrocarbon (Ah) receptor binding affinities. The results showed that 2,3,7,8-TCDD did not alter the IGF-I receptor mRNA levels or the KD values for binding of [125I]IGF-I to the IGF-I receptor in MCF-7 cells. However, 2,3,7,8-TCDD significantly decreased the number of IGF-I-induced IGF-I receptor binding sites and this may play a role in the growth-inhibitory properties of 2,3,7,8-TCDD and related compounds and in the 'cross-talk' between the two endocrine-response pathways.


Evidence that the stimulation by arginine vasopressin of the release of adrenocorticotropin from the ovine anterior pituitary involves the activation of protein kinase C. These studies were undertaken to evaluate the role of protein kinase C (PKC) in the regulation by arginine vasopressin (AVP) of adrenocorticotropin (ACTH) secretion from the ovine anterior pituitary. AVP caused the rapid translocation of PKC from the cytosol to the cell membrane in ovine anterior pituitary cells that was maximal at 5 min. This phenomenon, which is a known concomitant of C-kinase activation, was produced to a greater extent by phorbol 12-myristate 13-acetate (PMA) but not by corticotropin-releasing factor (CRF). To determine whether AVP activated corticotrope PKC, we assessed the ability of three different PKC inhibitors (H-7, sphingosine, and retinal) to modify basal, AVP-, PMA-, and CRF-stimulated ACTH release. In addition to inhibiting the in vitro activity of purified PKC, each compound also caused in vitro inhibition of the protein kinase A (PKA) catalytic subunit, indicating that none could be considered to be a specific inhibitor of PKC and the PKA catalytic subunit. As determined by the mean IC50 values required for the in vitro inhibition of PKC and the PKA catalytic subunit, sphingosine was judged to be the most selective and H-7 the least selective PKC inhibitor. A 4 h exposure to each inhibitor caused a dose-dependent increase in basal ACTH release and attenuation of both AVP- and PMA-stimulated ACTH release. H-7 and retinal, in concentrations that caused a 20-50% inhibition of PKA, also attenuated CRF-stimulated ACTH release; however, this effect was not observed with sphingosine in concentrations that caused only a 10-20% inhibition of PKA. We conclude that: (1) AVP causes the direct activation of PKC in the ovine anterior pituitary and that C kinase activation is important in mediating the effect of AVP on ACTH release; (2) the finding that inhibition of PKC elevates ACTH suggests that basal ACTH secretion is also partly regulated by PKC; (3) since CRF does not cause PKC translocation in ovine anterior pituitary cells, it is unlikely that PKC plays a physiological role in the action of CRF on the corticotrope; (4) the finding that H-7 and retinal attenuate CRF-stimulated ACTH secretion suggests that CRF activates PKA in corticotropes.


[MDG1het and aurora--non-mobile retrotransposons of Drosophila melanogaster]. Non-mobile retrotransposons mdg1het and aurora localized in Drosophila melanogaster heterochromatin were studied. A novel retrotransposon aurora comprising 324 bp LTRs was revealed as a 5 kb insertion causing 5 bp duplication of integration site in the heterochromatic Stellate gene. All the aurora copies are immobilized in D. melanogaster heterochromatin and adjoining chromosome regions 40, 41C and 80BC. Mobile aurora copies were revealed in D. simulans euchromatin by in situ hybridization technique. A comparison of 2.5 kb sequence of immobile mdg1het (including a half of ORF2 and 3'-LTR) with the correspondent sequence of transposable mdg1 copy [9] allowed to conclude that evolution of mdg1 subfamilies occurred under the selective pressure for the ability to transpose. The time period passed since the aurora and mdg1 copies integrated in heterochromatin was roughly estimated via divergence extent between the left and right LTR; for aurora copy it is 0-0.15 Myr, and for mdg1het copies it is 0-0.7 Myr.


Estradiol regulates gonadotropin-releasing hormone receptor number, growth and inositol phosphate production in alpha T3-1 cells. Gonadal steroids act at the pituitary to regulate gonadotropin-releasing hormone (GnRH) receptor number and the responsiveness of gonadotropes to GnRH and can act at post-receptor sites to modulate Ca(2+)-mediated and protein kinase C-mediated signal-transducing pathways. However, such effects have been seen in the mixed cell population of primary cell cultures and may involve indirect effects on cells other than gonadotropes. Here, steroid effects on a recently described gonadotrope-derived cell line (alpha T3-1 cells) have been assessed. In these cells estradiol, progesterone, testosterone and corticosterone all exerted trophic effects. Estradiol increased [3H]thymidine incorporation with an EC50 of 10(-12) to 10(-11) M and this effect was blocked by keoxifene, an estrogen receptor antagonist. Estradiol also reduced binding of [125I]buserelin (EC50 approximately 10(-11) M), an effect which appears to reflect a reduction in GnRH receptor number rather than a change in Kd. Estradiol also shifted the dose-response curve for GnRH-stimulated inositol phosphate (IP) accumulation rightward, increasing the EC50 for this GnRH effect by approximately 20-fold. Accordingly estradiol acts directly upon alpha T3-1 cells not only to reduce GnRH receptor number, but also to reduce the efficiency of coupling of residual GnRH receptors to second messenger generation.


[Role of fructose-1-phosphate kinase in expression of PEP-synthase in Escherichia coli K-12]. Mutational damage of the fruK gene coding for fructose-1-phosphate kinase leads to 2-6-fold (depending on the strain) decrease in FEP synthase activity in Escherichia coli. The fruK mutants were unable to utilize lactate as well as fructose and fructose-1-phosphate, acquiring, in addition, sensitivity to mannose in their growth medium. Reversions back to FruK+ phenotype or introduction of an intact fruK allele resulted in restoration of both FEP synthase activity and the ability to grow on lactate.


RNase H-mediated inhibition of translation by antisense oligodeoxyribonucleotides: use of backbone modification to improve specificity. A sequence of the rabbit alpha-globin mRNA is the primary target for ODN1, an unmodified 15-nucleotide (nt) antisense oligodeoxyribonucleotide (oligo). ODN1 prevented in vitro translation of both alpha- and beta-globin mRNAs in wheat germ extract. Nine secondary sites exhibiting more than 60% complementarity with ODN1 were present in the beta-globin message. The ODN1 inhibition of beta-globin synthesis was shown to be mediated by RNase H cleavage of the beta-globin mRNA at three partially complementary sites. Sandwich-type oligos consisting of a stretch of unmodified nt with a few methylphosphonate residues at both 5' and 3' ends were derived from ODN1. We have demonstrated that one such analogue (ODN2), with five phosphodiester linkages in the central region, exhibited improved specificity for alpha-globin mRNA compared with the unmodified parent 15-mer, due to a reduced ability of RNase H to cleave beta-mRNA/ODN2 mismatched duplexes.


Reticuloendotheliosis virus long terminal repeat elements are efficient promoters in cells of various species and tissue origin, including human lymphoid cells. Promiscuous transcriptional activity of the reticuloendotheliosis virus (REV) long terminal repeat (LTR) was detected in transient expression assays using LTR-chloramphenicol acetyltransferase-encoding gene chimeras, and cells of diverse species and tissue type; levels of expression from two different REV LTRs correlate with reports of pathogenicity of the respective viruses in vivo. REVs do not encode a transactivator targeted to the viral LTR, and cells infected with Marek's disease virus, a herpesvirus with an overlapping host range, do not express factors that preferentially enhance expression from REV or avian sarcoma/leukemia virus LTRs. REV LTRs work efficiently in human lymphoid cells, and are viable alternatives to promoters commonly used for expression of cloned genes. They may also prove useful in the identification of new, ubiquitous cellular transcription factors.


Structure of the Drosophila melanogaster gene encoding cyclin A. A P element (PE)-induced Drosophila melanogaster mutation, hari, affects the formation of the bristle mechanosensory organ in the adult fly. In this mutation, the site of PE insertion is in the first intron of the gene (CycA) encoding cyclin A (CycA). In order to analyze the hari mutant at the molecular level, we cloned and sequenced the cDNA and genomic DNA encoding CycA. CycA has seven exons and six introns, and its transcription unit spans 6 kb. All exon-intron junctions are compatible with the GT/AG consensus. Results of primer extension analysis and RNase protection assay indicate that CycA has major and minor transcription start points (tsp). To our knowledge, this is the first report on the CycA genomic sequence from a multicellular organism.


Structure of the rat gene encoding the mitochondrial benzodiazepine receptor. The gene encoding the rat mitochondrial benzodiazepine receptor (MBR) was cloned and characterized. Hybridization of a previously cloned cDNA for MBR to genomic Southern blots indicated that the gene was probably present at one copy per haploid genome. Rapid amplification of cDNA ends with rat adrenal RNA was used to obtain 47 nt of additional sequence upstream from our previously cloned MBR cDNA proving to be a crucial step in cloning the first exon of this gene. The MBR gene is comprised of four exons spanning approx. 10 kb. The first intron, contained within a 8-kb stretch of this gene, is located within the 5'-untranslated sequence, whereas the remaining two introns are much shorter (641 and 854 bp) and interrupt the coding sequence. The third intron contains sequences homologous to rodent B1 repetitive elements and a novel sequence closely resembling part of a repetitive element belonging to the Alu family in humans. The transcription start point was mapped by S1 nuclease protection assays suggesting that the first exon is just 56 bp in length. The sequence upstream from this region contains three GC boxes but lacks other known consensus recognition sites for sequence-specific transcription factors.


A unique ascorbate peroxidase active component in the cyanobacterium Synechococcus PCC 7942 (R2). Ascorbate peroxidase active component (APAC) was purified and characterized in Synechococcus PCC 9742 (R2) cells. APAC was isolated from freshly harvested cells, by ion exchange chromatography on DEAE cellulose, ultrafiltration through a 3000 dalton cut off filter and high pressure liquid chromatography through a reversed phase C-18 column. APAC was found to be extremely stable to harsh treatments of boiling water for 30 min, acidification to pH 2.0 and proteolytic digestion. A close correlation between activity and iron content of APAC was observed throughout the purification steps. E.S.R. spectrum of APAC showed a resonance line at g = 4.3 in the oxidized from. Peroxide reduction by ascorbate decreased the E.S.R. signal, which reappeared upon reoxidation by H2O2. The affinities of APAC to H2O2 and ascorbate were high (0.38 mM and 0.2 mM, respectively). Amino acid composition analysis of APAC revealed the presence of glutamic acid:glycine:cysteine residues at 2:1:1 ratio.


An E.S.R. investigation of the reactive intermediate generated in the reaction between FeII and H2O2 in aqueous solution. Direct evidence for the formation of the hydroxyl radical. The technique of E.S.R. spectroscopy, when employed in conjunction with a continuous flow system, provides direct evidence for the nature of free radicals formed from organic substrates in the presence of FeII and H2O2 in aqueous solution. It is shown, both via the identification of hydroxyl-radical adducts to alkenes and via the observed site-selectivity of radical attack, that the hydroxyl radical is formed as the reactive intermediate in the presence of various chelators (e.g. EDTA, DTPA). This approach also allows the rate constants for the FeII-H2O2 reaction in the presence of the different chelates to be determined; values obtained are in reasonable agreement with most of those measured by other methods. Examples of radical oxidation (by FeIII) and reduction (by FeII) are revealed.


The role of superoxide anions in the establishment of an interferon-alpha-mediated antiviral state. It has been suggested that CuZn-superoxide dismutase (CuZnSOD) is required for the establishment of an interferon (IFN)-mediated antiviral state. To investigate this possibility further, a panel of 6 stably transfected HeLa clones, expressing CuZnSOD activity from 1.6 to 7.3 times the normal level, were treated with different concentrations of recombinant human interferon alpha A (rHuIFN-alpha A) followed by challenge with vesicular stomatitis virus (VSV). A biphasic response curve was generated (r = 0.87, p less than 0.025). Clones with up to 3-fold basal level CuZnSOD activity exhibited an inverse relationship between their ability to generate an IFN-alpha-mediated antiviral state and CuZnSOD activity: the higher the CuZnSOD activity, the lower the sensitivity to IFN-alpha and the more IFN-alpha required for antiviral defense. Clones with between 4 to 7.3 times higher CuZnSOD activity than the non-transfected HeLa control showed a direct relationship between the CuZnSOD activity and the sensitivity to IFN-alpha. Furthermore, in agreement with the results obtained with the SOD1-transfected HeLa cells with up to 3 times the basal SOD activity, fetal fibroblasts derived from SOD1-transgenic mouse strains, TgHS-229 and TgHS-218, which also express 3 times the basal CuZnSOD activity, required higher IFN-alpha to achieve 50% protection. These results suggest a possible role for superoxide anion in the establishment of IFN-mediated antiviral effect, especially in the dose-response region in which the inverse relationship between the generation of the IFN-alpha-mediated antiviral state and CuZnSOD activity was observed. To assess this possibility, allopurinol was used as a xanthine oxidase inhibitor and hydroxyl radical scavenger in the IFN-alpha-mediated antiviral assay. Addition of 3 mM allopurinol diminished the IFN-mediated antiviral effect by between 40 and 50% (p less than 0.01), and there was a reduction in superoxide generation (p less than 0.05). The degree of reduction caused by allopurinol treatment was higher at an IFN-alpha concentration of 10 U/ml than at 100 U/ml, and there was no correlation between CuZnSOD activity and the degree of reduction. To establish further the role of superoxide as an antiviral agent, paraquat was used as a superoxide generator in the absence of IFN-alpha in the antiviral assay. Although paraquat at high concentrations is toxic to the cells, it actually showed a protective effect against VSV infection, and an inverse relationship (r = 0.79, r less than 0.025) between cell survival and CuZnSOD activity was observed with 150 mM paraquat treatment.(ABSTRACT TRUNCATED AT 400 WORDS)