The Drosophila retinoid X receptor homolog ultraspiracle functions in both female reproduction and eye morphogenesis. Ultraspiracle (usp) encodes the Drosophila cognate of RXR, the human retinoid X receptor. To examine how RXR subfamily members function in development, we have undertaken a phenotypic analysis of usp mutants. usp is required at multiple stages of development for functions that occur in a wide variety of tissues. usp is required in the eye-antennal imaginal disc for normal eye morphogenesis and in the somatic and germline tissues of adult females for fertilization, eggshell morphogenesis and embryonic development. An unusual sunken eye phenotype with marked ventral-dorsal polarity appears to be caused by a lack of usp function in the imaginal disc cells that reside between the eye and antennal anlage. The usp functions include cell autonomous and non-cell autonomous components, suggesting that usp controls the production of factors important for both cell-cell communication and cellular differentiation. These usp signalling pathways have mechanistic parallels to steroid and retinoid action in developing vertebrate tissues.


Spatial and temporal expression of the I factor during oogenesis in Drosophila melanogaster. The I factor is a functional non-viral retrotransposon, or LINE, from Drosophila melanogaster. Its mobility is associated with the I-R hybrid dysgenesis. In order to study the expression pattern of this LINE in vivo, a translational fusion between the first ORF of the I factor and the lacZ gene of Escherichia coli has been carried out and introduced in the genome of reactive (R) flies. Homozygous transgenic Drosophila lines have been established and analysed. ORF1 expression is limited to germ-line cells (nurse cells and oocyte) between stage 2 and 10 of oogenesis. No somatic expression is found. Position effects may limit the level of expression of a given transgene but do not modify its basic pattern of expression during the development of the fly. This reproducible control demonstrates both that I factor is driven by its own promoter, probably the internal one suggested by Mizrokhi et al. (Mizrokhi, L.J., Georgevia, S.G. and Ilying, Y.V. (1988). Cell 54, 685-691), and that tissue-specific regulatory sequences are present in the 5' untranslated part of the I factor. The nuclear localization of the fusion protein reveals the presence of nuclear localization signals (NLS) in the ORF1-encoded protein correlating with the possible structural and/or regulatory role of this protein. This expression is restricted to dysgenic and reactive females, and is similar in the two conditions. All the results obtained in this work suggest that I factor transposition occurs as a meiotic event, between stage 2 and 10 of the oogenesis and is regulated at the transcriptional level. It also appears that our transgene is an efficient marker to follow I factor expression.


Overexpression of Dd PK2 protein kinase causes rapid development and affects the intracellular cAMP pathway of Dictyostelium discoideum. The Dd PK2 gene codes for a putative protein of 648 amino acids with a C-terminal half sharing high homology with protein kinase A catalytic subunits from other organisms. In order to find out more about the physiological role of the Dd PK2 kinase, its gene, and a version having a frame shift mutation in the middle of the catalytic region, were overexpressed in developing Dictyostelium cells. Both the intact gene (K-) and the frame shift mutant (Kdel-) caused rapid development with spores formed in 16-18 hours compared to the 24 hours required by their parent. This result was confirmed by the pattern of expression of some developmentally regulated genes. Other rapid developing strains (rde) are activated in the cAMP second messenger system. Both K- and Kdel-containing strains have lower cAMP levels than the parental strain during late development, thus resembling rdeC mutants. K-cells (but not Kdel-cells) produced bizarre fruiting bodies with many prostrate forms. The parallel with rde mutants was confirmed by demonstrating that K-cells are able to form spores in submerged monolayer culture. Furthermore, K-cells have about four times more protein kinase A (cAPK) activity than wild-type cells. These results indicate that the N-terminal domain of Dd PK2 is sufficient to influence cAMP levels and to provoke rapid development, whereas kinase activity seems to be required for the sporogenous phenotype. The association between elevated cAPK and Dd PK2 overexpression phenotype further indicates a role for cAPK in the formation of spores.


Local anesthesia in rhinoplasty: a new twist? Twenty volunteers were asked to compare pain upon injection during septorhinoplasty using buffered versus unbuffered local anesthetics. The concentration of the buffer was one part sodium bicarbonate to five parts local. The surgeons performing the operation were asked to identify any difference in hemostasis or duration of anesthesia. Eighteen of twenty patients found the buffered anesthetic to be less painful and better tolerated. No difference in hemostasis or duration of action was noted between the buffered or unbuffered solution, however, faster onset of action was noted with the buffered solution. The addition of sodium bicarbonate as a buffering agent to the local anesthetics lidocaine and bupivacaine can significantly reduce pain upon injection. A solution of 5cc 2% lidocaine with 1:100,000 epinephrine, 5cc 0.25% bupivacaine with 1:200,000 epinephrine, and 2cc of 7.5% sodium bicarbonate mixed just prior to injection is a safe, effective, less painful local anesthetic with rapid onset of action and full efficacy.


Circadian timekeeping during pregnancy: endogenous phase relationships between maternal plasma hormones and the maternal body temperature rhythm in pregnant rhesus monkeys. Nine pregnant rhesus monkeys maintained in constant low level lighting (5 Lux) from 56-80 days gestation (dGA) onward were studied to assess the presence or absence of circadian maternal body temperature and maternal plasma hormone variations. Maternal arterial blood samples were taken every 4 h in six monkeys (105-120 dGA) for 48-56 h and every 2 h in three monkeys (108-115 dGA) for 60 h. Maternal intraabdominal temperature was recorded continuously. Cosinor analysis was used to determine circadian rhythmicity. Individual endogenous timekeeping was demonstrated by 1) free-running circadian variations in maternal body temperature in all nine animals, 2) consistent internal acrophase relationships between the maternal body temperature and maternal plasma cortisol rhythm, and 3) idiosyncrasy of the temporal relationship of detectable rhythms to the external environment in individual animals. Only one animal had a significant maternal plasma ACTH rhythm, whereas the presence of 24-h variations in the other hormones varied in individual animals. The mean +/- SD acrophase profiles in hours from the temperature acrophase in those animals who showed significant rhythms were 19.7 +/- 0.6 (n = 8) for cortisol, 19.4 +/- 2.4 (n = 6) for dehydroepiandrosterone sulfate, 8.3 +/- 1.1 (n = 6) for progesterone, and 18.9 +/- 1.6 (n = 3) for estradiol. We conclude that 24-h variations in maternal plasma hormones are truly endogenously generated and not passively dependent on the light-dark cycle. The maternal circadian system regulates the 24 h temporal organization of endogenous plasma hormone variations. We hypothesize that rhythms in cortisol, dehydroepiandrosterone sulfate, progesterone, and estradiol during pregnancy are directly or indirectly governed by the maternal hypothalamus via the circadian oscillatory output of the maternal adrenal.


Increased cellular cholesterol upregulates high density lipoprotein binding to rat luteal cells. Rat luteal cells preferentially utilize cholesterol derived from high density lipoproteins (HDL) as a substrate for steroid hormone synthesis. The uptake of cholesterol from HDL by these cells is in contrast to nonsteroidogenic cells, which export cholesterol to HDL. A previous study demonstrated that HDL binding to luteal cell membranes was increased in conjunction with in vivo cholesterol depletion or cholesterol loading of the ovary induced by pharmacological agents. These results suggest a biphasic regulation of the HDL receptor in luteinized rat ovaries. In the present studies, the in vitro regulation of HDL binding in rat luteal cells by increased intracellular cholesterol was examined. Cultured luteal cells were incubated with increasing doses of low density lipoproteins (LDL) for 2 days after which the cellular sterol content and the effects on progesterone production and HDL binding were measured. As expected, the LDL treatment increased total cellular sterol content in a dose-dependent manner, resulting in a 2.1-fold increase over control at a dose of 1 mg LDL/ml. Increased cellular cholesterol was accompanied by a comparable increase in progesterone secretion. These results suggest that exogenous cholesterol was utilized by these cells. The LDL treatment also increased the binding of HDL to the cells in a dose-dependent manner to a maximum of 2.2-fold over control. The effect of increased cellular sterol on HDL binding was also examined using a more polar cholesterol derivative, 25-hydroxycholesterol. Cells were cultured for 2 days in media containing 0.3-40 micrograms/ml 25-hydroxycholesterol in the presence of 100 micrograms/ml aminoglutethimide, an inhibitor of cholesterol metabolism. The HDL binding to luteal cells exhibited dose-dependent up-regulation by 25-hydroxycholesterol with a 5.8-fold increase in binding at the maximum dose tested. Equilibrium binding studies using cells treated with 10 micrograms/ml 25-hydroxycholesterol revealed a 2.1-fold increase in the number of HDL binding sites on the luteal cells without affecting the binding affinity. From the results of this study, it is concluded that HDL binding in rat luteal cells is up-regulated by an increase in the intracellular cholesterol level.


The effect of "binge" ethanol exposure on growth hormone and prolactin gene expression and secretion. The effects of ethanol (EtOH) on GH and PRL have been previously explored, and a dicotomy in results noted. While serum GH levels appear to fall after EtOH exposure, PRL levels rise. We have attempted to expand these studies by examining the impact of acute or "binge" EtOH in vivo on GH and PRL synthesis and secretion. At 0.5, 1.5, and 3.0 h after one dose of ip EtOH, serum GH levels fell significantly compared with those seen in saline-injected controls. This correlated with a fall in GH mRNA levels, but no change in pituitary GH content. Conversely, serum PRL levels rose significantly, while the mRNA for PRL decreased by approximately 20%. There was no change in pituitary PRL content. Interestingly, the mRNA for pit-1 (GHF-1), a transcription factor important to both GH and PRL gene expression, was unchanged at any time point. Despite the fall in GH and PRL mRNA levels, the pituitary cAMP content was markedly elevated at 0.5 h, with no change at any other time point. In summary, acute EtOH exposure in vivo appears to dampen both GH and PRL synthesis, while serum levels behave dissimilarily. Possible explanations for these findings are discussed.


Evidence for further heterogeneity of the receptors for neuropeptide-Y and peptide-YY in tumor cell lines derived from neural crest. The expression and structure of the receptors for neuropeptide-Y (NPY) and peptide-YY (PYY) were studied in 16 human and rodent tumor cell lines derived from the neural crest by ligand binding and cross-linking techniques using [125I]Bolton-Hunter-NPY, [125I]PYY, and various forms of monoiodinated NPY and PYY. Although NPY-binding sites were observed in most of the tumor cells, PYY-binding sites were found only on the human neuroblastoma cell lines SMS-MSN, SMS-KAN, SK-N-MC, and MC-IXC and the human Ewing's sarcoma cell line SK-ES. The differential labeling of the NPY/PYY receptors on these cell lines suggests that the NPY/PYY receptors are more heterogeneous than previously described as the Y1, Y2, and Y3 receptor subtypes. Cross-linking studies demonstrate that the Y1 and Y2 receptors for NPY/PYY are structurally different (mol wt, 70 and 50 kilodaltons, respectively) and that the 70- and 50-kilodalton receptor proteins are coexpressed in certain tumor cell lines. This could explain at least in part why cell lines show a relative specificity for Y1/Y2 classification, observed as the inhibition by both C-terminal fragments and Y1-specific analogs on the NPY/PYY binding to membrane receptors. Collectively, the present study suggests further heterogeneity of the NPY/PYY receptors and the existence of multiple receptor proteins in the tumor cell lines derived from the neural crest.


Follicle-stimulating hormone-regulated Sertoli cell proteins SCc1 and SCc2 are phosphorylated and mitochondrially associated. Sertoli cell intracellular protein 1 (SCc1) and 2 (SCc2) are polypeptides found in rat Sertoli cell cultures incubated with either FSH or (Bu)2cAMP. They were first identified in [35S]methionine-labeled Sertoli cell lysates using two-dimensional gel electrophoresis. Here we extend these observations by showing that SCc1 and SCc2 are present in rat seminiferous tubules, ovaries, and granulosa cells incubated with either FSH or (Bu)2cAMP and in testicular peritubular cells incubated with (Bu)2cAMP. Peritubular cells do not, however, respond to FSH with the production of SCc1 and SCc2. Peptide mapping with N-chlorosuccinimide revealed that SCc1 and SCc2 have similar cleavage patterns, suggesting a common primary amino acid sequence that is modified posttranslationally. Metabolic labeling with [32P]orthophosphate provided direct evidence that SCc1 and SCc2 are phosphoproteins. A shift in mobility of SCc1 and SCc2 toward the basic region of the gel to positions designated SCc1' and SCc2' occurred when cell lysates were treated with alkaline phosphatase before electrophoresis, providing additional evidence that SCc1 and SCc2 are phosphoproteins. SCc1 and SCc2 are also shown to be mitochondrially-associated in the Sertoli cell. Peptide maps of SCc1, SCc2, SCc1', and SCc2' obtained by treatment with alpha-chymotrypsin, are identical to proteolytic maps of proteins pp30', p30, and pp30 from adrenocortical cells. SCc1, SCc2, SCc1', and SCc2' are homologous with regard to their regulated expression, electrophoretic mobility, and mitochondrial localization to the adrenal proteins pp30' and pp30 as well as a series of 30 kilodalton proteins from MA-10 Leydig tumor cells. Both the adrenal cell proteins and the Leydig tumor cell proteins are thought to participate in cholesterol transport to the inner mitochondrial membrane, providing substrate for the cholesterol side-chain cleavage enzyme complex, an activity which the Sertoli cell does not perform, suggesting that alternative functions must be sought for SCc1 and SCc2 in Sertoli cells.


The effects of prostaglandin E2, parathyroid hormone, and epidermal growth factor on mitogenesis, signaling, and primary response genes in UMR 106-01 osteoblast-like cells. Prostaglandin E2 (PGE2), PTH, and epidermal growth factor (EGF) are potent regulators of osteoblast proliferation. In UMR 106-01 rat osteosarcoma cells with osteoblast-like features, PGE2 and PTH inhibit, while EGF stimulates, mitogenesis. Both PGE2 and PTH increase intracellular cAMP levels, cytosolic calcium, and inositol phosphate turnover. In a variety of cell types, EGF mediates its effects in part via activation of receptor protein-tyrosine kinase and other protein kinases, such as protein kinase-C. The nuclear mechanisms of PGE2, PTH, and EGF regulation of osteoblast proliferation are unknown. Accordingly, we have examined the effects of these agents on mitogenesis, second messenger generation, and primary response genes, which may link second messenger activation to subsequent alterations in gene expression. Northern blot analysis of mRNA from UMR 106-01 cells treated for 3 h with 2 microM PGE2, 10 nM PTH, or 10 ng/ml EGF in the presence of cycloheximide demonstrated that all three agents induced the expression of c-fos and c-jun mRNA. In contrast, only EGF stimulated cellular proliferation and induced Egr-1 mRNA. Also, unlike PGE2 and PTH, EGF did not increase intracellular cAMP levels. c-fos mRNA was induced by treatment with 50 ng/ml tetradecanoyl phorbol acetate or by 40 ng/ml forskolin, while induction of Egr-1 mRNA was stimulated by treatment with tetradecanoyl phorbol acetate, but not forskolin. Thus, EGF signal transduction differs from that of PGE2 and PTH in UMR 106-01 osteoblast-like cells, in that EGF does not stimulate the protein kinase-A second messenger system, but causes activation of Egr-1, a primary response gene that may play a role in the mitogenic effect of EGF.


Involvement of nitric oxide in growth hormone (GH)-releasing hormone-induced GH secretion in rat pituitary cells. The involvement of nitric oxide (NO) in human GH-releasing hormone (hGHRH)-induced GH secretion was studied with freshly dissociated male rat pituitary cells. The cells were packed in a column of Bio-Gel-P2 and continuously perifused at 37 C. Hemoglobin (Hb; 10 microM), which is known to strongly bind NO, potentiated 0.01, 0.1, and 1 nM hGHRH-induced GH secretion by 73%, 52%, and 39%, respectively, without affecting the basal secretion of GH. As reported previously, 1-nM or higher concentrations of hGHRH elicit an increase in GH secretion during the application of hGHRH (on-response) and also a transient increase after the cessation of hGHRH application (off-response). It was found that Hb potentiated only the off-response in 1 nM hGHRH-induced GH secretion, and the same concentration of Hb had no effect on 10 nM hGHRH-induced GH secretion. N-Methyl-L-arginine (MeArg; 500 microM), a competitive inhibitor of NO synthase, also potentiated both the on- and off-responses of 1 nM hGHRH-induced GH secretion by 39% without affecting basal GH secretion. Since cAMP is thought to be an intracellular messenger of hGHRH action, the effects of Hb and MeArg on 1 mM (Bu)2AMP-induced GH secretion were examined. Their actions were found to be greater than those in hGHRH-induced GH secretion. Excess K+ (15 and 50 mM)-induced GH secretion, which does not involve cAMP, however, was not affected by either Hb or MeArg. In contrast, 3 mM sodium nitroprusside, which releases NO, suppressed the 1 nM hGHRH-induced off-response by 18%. The same concentration of sodium nitroprusside had no effect on excess K(+)-induced GH secretion. The effect of 8-bromo-cGMP on hGHRH-induced GH secretion was also examined, since NO is thought to exert its action through cGMP by activating guanylate cyclase in neural tissue. The application of 8-bromo-cGMP, however, did not affect 1 nM hGHRH-induced GH secretion. These observations suggest that hGHRH stimulates the synthesis of NO at least partly through cAMP, thereby partially inhibiting hGHRH-induced GH secretion.


The role of protein kinase-A activity in the evaluation of agonist/antagonist properties of analogs of parathyroid hormone-related protein in opossum kidney cells. Structural modifications of the amino-terminal region of PTH resulted in the generation of PTH analogs with potent antagonist activities and markedly reduced agonist activities. Further development of these structure-function relationships to modifications of the sequence of the PTH-related protein (PTHrP) resulted in PTH/PTHrP antagonist analogs with increased antagonist activity and little if any agonist effects. Since studies from our laboratory have shown that measurement of protein kinase-A activity appears to be a more sensitive index of the actions of PTH than measurements of total cAMP, the present studies were designed to examine the effects of a series of PTHrP-based peptides for agonist/antagonist effects in opossum kidney (OK) cells. The results show that PTHrP-(7-34)NH2, which does not increase cAMP, displays agonist activity in terms of increasing protein kinase-A activity. Furthermore, [Leu11,D-Trp12]PTHrP-(7-34)NH2 and [D-Trp12]PTHrP-(7-34)NH2, which appear to be effective antagonists of rat PTH-(1-34)-stimulated cAMP generation, were less effective in antagonizing the effects on protein kinase-A and only [Leu11,D-Trp12] PTHrP-(7-34)NH2 appeared to exhibit any antagonist activity. The Ki for [Leu11,D-Trp1/]PTHrP-(7-34)NH2 to antagonize the stimulatory effect of 1 nM rat PTH-(1-34) was easily demonstrable by measurements of cAMP, but could not be demonstrated using the assay of protein kinase-A activity. These data underscore the observation that measurement of protein kinase-A is a more sensitive index of the effects of PTH than measurements of cAMP and that significant agonist activity on the cAMP/protein kinase pathway cannot be excluded without measurements of protein kinase-A activity.


Effect of epidermal growth factor on inhibin secretion in human placental cell culture. The effect of epidermal growth factor (EGF) on inhibin secretion was investigated in a primary culture of human placental cells. Dissociated cells were cultured with EGF, FSH, 8-Br-cAMP, and two agents known to increase intracellular cAMP. Inhibin level in the culture medium was measured by immunoenzymatic assay. Addition of EGF (0.1-1000 ng/ml) in the cell culture induced a dose-dependent increase of inhibin levels in the medium after 2 days of culture. Greater response of placental cells to EGF in the inhibin secretion occurred at the doses of 10-1000 ng/ml, where inhibin levels in the medium increased by 84.9-111.5% compared to the control (P < 0.01). FSH stimulated the inhibin secretion in the placental cells. EGF combined with FSH resulted in a greater response of placental cells in inhibin secretion. Addition of FSH (30 ng/ml) and EGF (0.1-1000 ng/ml) in the culture induced inhibin levels significantly higher than that of either FSH alone or EGF alone (P < 0.01). The effect of EGF on inhibin secretion was closely correlated with the seeding density of trophoblasts and the time course of culture. Obvious effect of EGF was found at the number of 1-2 x 10(6) cells per well and after 36-48 h of culture. Addition of 8-Br-cAMP, cholera toxin, or forskolin in the culture increased the inhibin levels more than 6-fold, 5-fold, and 2-fold, compared to the controls, respectively. When EGF combined with one of these agents was added in the culture, the inhibin in the medium increased to a level higher than those with the individual agents alone. EGF resulted in an increase in basal and cAMP induced human CG secretions in the trophoblasts in a similar manner as in the inhibin secretion. However, the effect of EGF on the proliferation of trophoblasts was not observed by measurements of the cell growth with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and DNA content in the cells with fluorescence spectrophotometry. Morphological study showed that EGF induced trophoblasts to differentiate and form syncytium. These data suggest that EGF stimulates inhibin secretion in human placental cells in vitro. EGF and its interaction with other hormones or growth factors may play an important role in the complicated hormonal regulation during human pregnancy.


Evolution of metabolic and functional derangements of pancreatic islets in phosphate depletion. Phosphate depletion (PD) causes a rise in basal level of cytosolic calcium ([Ca2+]i) of pancreatic islets, a decrease in their basal and stimulated ATP content, a reduction in the maximum velocity (Vmax) of Ca2+ adenosine triphosphatase (ATPase) and Na(+)-K+ ATPase, impaired glucose-induced calcium signal and decreased glucose-induced insulin secretion. The sequence of events that lead to these derangements during the evolution of PD are not defined. The present study examined this issue by measuring the metabolic and functional profile of pancreatic islets weekly during the evolution of PD over a period of 6 weeks, and whether phosphate repletion reverses these abnormalities. The results show that initial abnormalities are a rise in Vmax of Ca2+ ATPase and modest rise in basal [Ca2+]i. This was followed by a fall in basal and stimulated ATP content. With the fall in ATP content, the Vmax of Ca2+ ATPase and Na(+)-K+ ATPase decreases and the rise in [Ca2+]i becomes more pronounced. A decrease in glucose-induced insulin secretion becomes evident with the fall in ATP, the decrease in glucose-induced calcium signal, and/or delta[Ca2+]i/basal[Ca2+]i. All functional and metabolic derangements of the pancreatic islets returned to normal after phosphate repletion. Taken together, our data are consistent with the notion that PD is associated with an initial increase in calcium influx into the islets. This is followed by modest but significant rise in [Ca2+]i which, in turn, would inhibit mitochondrial oxidation and ATP generation leading to a decrease in ATP content. The latter compromises the activity of Ca2+ ATPase and Na(+)-K+ ATPase which are involved, directly or indirectly, in calcium extrusion out of the islets. The increased influx of calcium combined with decreased calcium extrusion is followed by a further rise in basal levels of [Ca2+]i. This sequence of events continues until a steady state is reached and is characterized by reduced basal and stimulated ATP content, reduced Vmax of Ca2+ ATPase and Na(+)-K+ ATPase and elevated basal level of [Ca2+]i. Phosphate repletion reverses all these abnormalities.


Angiotensin II potentiates agonist-induced 3',5'-cyclic adenosine monophosphate production by cultured bovine adrenal cells through protein kinase C and calmodulin pathways. Interactions between signal transducing systems may be important in the integrated control of cellular processes in basal and hormonally regulated cells. The cultured bovine adrenal fasciculata cell provides a model to study the interactions between the cAMP and calcium-sensitive phospholipid dependent protein kinase C. In this study, angiotensin II (A-II) and phorbol ester (PMA) potentiated the stimulatory actions of ACTH in a dose-dependent manner on cAMP production. At maximal concentrations, A-II and PMA also potentiated the effects of cholera toxin and forskolin on cAMP production. Both staurosporine, a protein kinase C inhibitor, and desensitization of protein kinase C by a 24-h pretreatment with PMA blunted the effect of PMA, but only partially inhibited (34%) the effect of A-II. Neither nifedipine, a specific calcium channel antagonist, nor pretreatment of cells with pertussis toxin modified the amplifying effects of A-II or PMA. In contrast, trifluoperazine, a calmodulin inhibitor, reduced the potentiating effect of A-II by about 35%, but association with staurosporine blunted its effects. Moreover, the steroidogenic effects of ACTH plus A-II were more than additive, but this synergism was blunted in the presence of both inhibitors. In conclusion, PMA and A-II potentiated agonist-induced cAMP production by bovine adrenal fasciculata cells. The data suggest that the effects of PMA were mediated exclusively by protein kinase C, whereas those of A-II were mediated by both protein kinase C and calmodulin.


1,25-Dihydroxyvitamin D3 in the Atlantic cod: plasma levels, a plasma binding component, and organ distribution of a high affinity receptor. Physiological studies of the Atlantic cod, Gadus morhua, have suggested a role for the vitamin D3 system in this marine teleost similar to that in other vertebrates. Accordingly, the present study was carried out to assess the plasma concentrations of vitamin D3, 25-hydroxyvitamin D3 (25OHD3), and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in this fish. Additionally, the presence of binding proteins in plasma and target-specific tissue receptors for these vitamin D3 metabolites was studied in organs normally associated with calcium regulation. Plasma levels of 25-OHD3 (undetectable to 148 pg/ml; n = 5) were comparatively low (20-30 ng/ml), whereas the levels of vitamin D3 (approximately 30 ng/ml) and 1,25-(OH)2D3 (approximately 50 pg/ml) were comparable to levels reported in higher vertebrates. Cod plasma contained a protein that bound both 25OHD3 and 1,25-(OH)2D3. This plasma binding protein revealed low affinity for 25OHD3, did not bind G-actin, and had a sedimentation coefficient of 3.4S. High affinity 1,25-(OH)2D3 receptors [Kd, 1.02 +/- 0.36 (n = 6), 1.02 +/- 0.3 (n = 5), and 0.95 +/- 0.51 (n = 5) nM; mean +/- SEM] were found in high salt cytosols from intestine, liver, and gills, respectively, and had sedimentation coefficients (3.6-3.8S in 0.3 M KCl sucrose gradients) similar to those in higher vertebrates. No specific 1,25-(OH)2D3 binding was found in kidney, ultimobranchial glands, corpuscles of Stannius, or bone. The finding of significant quantities of 1,25-(OH)2D3 in the plasma, the presence of plasma binding proteins that bind this seco-steroid, and the localization of specific high affinity receptors for this metabolite in calcium regulatory tissues in teleosts are all consistent with a physiological role for the vitamin D3 system in the calcium regulation of the cod.


Free fatty acids activate the hypothalamic-pituitary-adrenocortical axis in rats. Intravenous administration of Intralipid 10% increases blood levels of essential free fatty acids. In rats and man, this is associated with an inhibition of GH secretion from the anterior pituitary. Because GH is lipolytic, the inhibition of its secretion may represent a negative feedback action of the fats on pituitary sensitivity to GH-releasing hormone. Since corticosterone, the final secretory product of the rat hypothalamic-pituitary-adrenocortical (HPA) axis, is also lipolytic, we tested the hypothesis that FFA would inhibit the HPA axis. Rats were cannulated via the jugular vein and infused with different doses of heparin-Intralipid 10% or heparin-saline; sequential blood samples were obtained and analyzed for ACTH, corticosterone, FFA, and glucose. Intralipid at 2.85 ml/kg increased plasma FFA to over 3 meq/liter by 15 min, with a return to baseline by 60-90 min. There was no effect of the infusion on plasma osmolarity or pH. At 60 min, plasma ACTH levels were significantly elevated to over 1500 pg/ml in Intralipid-infused rats, but were unchanged in saline controls. This dose of Intralipid increased corticosterone levels by nearly 20-fold at 120 min. At 180 min, corticosterone levels were still significantly greater than those in saline controls. Lower doses of Intralipid also significantly elevated both FFA and corticosterone levels, but by 180 min, levels of both were similar to those in controls. The effects of Intralipid on corticosterone secretion could not be attributed to the presence of glycerol in the suspension, since glycerol infusions had no significant effect on steroid levels compared to those in saline controls. In dexamethasone-pretreated rats, there was no significant rise in plasma corticosterone after either of two Intralipid doses, suggesting that the action of Intralipid was at a site within the HPA axis above the adrenal gland. This finding also suggested that the high steroid levels after Intralipid treatment were not due to interference with the corticosterone RIA. This was verified by the finding that there was no increase in plasma immunoreactive corticosterone after Intralipid infusion into adrenalectomized rats. Intralipid also caused an increase in plasma glucose levels that was first significant at 60 min and declined to baseline by 180 min, possibly reflecting increased autonomic activity or peripheral insensitivity to insulin. The results suggest that high circulating FFA levels activate, rather than inhibit, the HPA axis in rats. Since stress activates glucocorticoid production and increases FFA levels due to lipolysis, it is possible that FFA and the HPA axis constitute a previously unrecognized positive feedback loop.(ABSTRACT TRUNCATED AT 400 WORDS)


Differential response of preadipocytes and adipocytes to prostacyclin and prostaglandin E2: physiological implications. The major prostaglandins (PGs) locally produced in adipose tissue both in rodent and man are PGE2 and prostacyclin (PGI2). We have recently described PGI2 as an autocrine promoter and/or amplifier of terminal differentiation of cultured preadipocytes in several species. The effectiveness and specificity of PGI2 as an adipogenic agent are related to its ability to induce in preadipocytes intracellular increases of both cAMP and free calcium. Moreover, PGs of the E series are well known to exert an antilipolytic effect in mature adipocytes. These observations have prompted us to address two questions of physiological interest: 1) Is PGI2 still able to increase cAMP in differentiated adipocytes, behaving thus as a lipolytic agent, and 2) Is PGE2 able to negatively modulate cAMP production in adipose precursor cells, behaving thus as a counteracting effector of PGI2 action? Our results, with respect to cAMP production and/or lipolysis and antilipolysis, demonstrate clearly that in adipose tissue of both rat and man, PGI2 exclusively affects adipose precursor cells whereas PGE2 exclusively affects adipocytes. We propose a model of concerted action for both PGs in the development of adipose tissue mass, PGI2 behaving as an adipogenic-hyperplastic effector and PGE2 as an antilipolytic-hypertrophic effector.


Studies on the mechanism of the novel stimulatory effect of angiotensin-II on adenylate cyclase in rat fetal skin fibroblasts. In contrast to other systems in which angiotensin-II (AII) inhibits adenylyl cyclase, in fetal skin fibroblasts the peptide stimulates cAMP accumulation. The mechanism of this novel effect was studied by analysis of the actions of AII and other regulators on the adenylyl cyclase system in cultured cells. In the presence of phosphodiesterase inhibitors, AII, isoproterenol (ISO), choleratoxin (CTx), and forskolin (Fk) stimulated cAMP accumulation by 2.0 +/- 0.26-, 26 +/- 0.9-, 75 +/- 5.6-, and 88 +/- 3.3-fold, respectively. AII potentiated the stimulatory effect of ISO and CTx by 1.5 +/- 0.1- and 1.25 +/- 0.03-fold, respectively, but had no effect on that of Fk. Preincubation of the cells with PTx did not prevent the stimulatory effect of AII on basal and ISO- and CTx-stimulated cAMP, indicating that the effect of AII was not due to interaction with Gi. Unexpectedly, pretreatment of the cells with PTx for 18 h inhibited cAMP production stimulated by ISO and Fk. Similar inhibition by PTx was observed in adult rat skin fibroblasts, but not in adult human fibroblasts, in which pretreatment with PTx resulted in potentiation of Fk-stimulated cAMP production. ADP ribosylation studies showed that the optical density of the band corresponding to Gs was less than 20% that of Gi and Go in rat fetal cells, suggesting that excess release of the beta-gamma-subunit is responsible for the inhibition of cAMP production by PTx. However, immunoblot analysis of G-proteins showed that the content of Gs alpha was similar to that of Gi alpha and Go alpha in rat and human, fetal and adult cells. In contrast to the effect in intact cells, AII had no effect on basal or stimulated adenylyl cyclase activity in cell homogenates, suggesting that the stimulatory effect observed in intact cells is indirect. The stimulatory action of AII on cAMP production was not blocked by indomethacin, indicating that the effect is not mediated by prostaglandin formation. The stimulation of cAMP by AII was mimicked by 10-min incubation with phorbol 12-myristate 13-acetate (PMA), and prevented after cellular protein kinase-C (PKC) depletion by 4- or 6-h preincubation with PMA. However, the stimulation was not prevented by the PKC inhibitors staurosporine and H7 or 24-h preincubation with PMA, suggesting that the effect is not mediated by a traditional PKC-dependent mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)


Effects of gonadectomy and sex hormone therapy on the endotoxin-stimulated hypothalamo-pituitary-adrenal axis: evidence for a neuroendocrine-immunological sexual dimorphism. Bacterial lipopolysaccharide (LPS) stimulates the hypothalamo-pituitary-adrenal axis by a mechanism involving the release of cytokines, which activate the CRH-ACTH system and, as a result, increase glucocorticoid secretion. In the present study we investigated the possibility that endogenous sex hormones modulate the in vivo endotoxin-stimulated adrenal and immune responses in adult BALB/c mice. In preliminary experiments we determined that the maximal glucocorticoid release in response to LPS (50 micrograms, ip) administration was reached 2 h after treatment. The endotoxin effect on the adrenal and immune responses was then tested in male, randomly cycling female, 20-day-gonadectomized and 20-day-gonadectomized mice treated with either testosterone or estradiol. In addition, in vitro experiments were performed to determine whether 1) LPS exerts any direct effect on basal and ACTH-stimulated corticosterone release, and 2) adrenal function is influenced by bilateral gonadectomy and sex steroid therapy. Our results indicate that 1) randomly cycling female mice have significantly more pronounced corticosterone secretion than males 2 h after endotoxin injection, although the tumor necrosis factor responses were similar; 2) the response of the hypothalamo-pituitary-adrenal axis to endotoxin stimulation in female mice was invariable throughout the different stages of the normal estrous cycle; 3) gonadectomy leads to enhanced (P < 0.05) adrenal and immune responses to LPS stimulation compared to the responses in shams; 4) the endotoxin-elicited adrenal and immune overresponses observed in gonadectomized mice are reversed by testosterone treatment, regardless of sex; 5) LPS does not directly modify spontaneous and ACTH-stimulated adrenal corticosterone secretion; and 6) gonadectomy alone or combined with sex steroid therapy does not increase the in vitro adrenal response to ACTH stimulation. Our findings further suggest an evident neuroendocrine-immunological sexual dimorphism during the acute phase of inflammatory processes.


Cell specific distribution of LH/hCG receptor messenger ribonucleic acid in rat testicular Leydig cells. By using four different cell isolation procedures, we previously identified two morphologically and biochemically distinct Leydig cell populations in rat testis. The light cells were vacuolated and bound 125I-labeled human choriogonadotropin (hCG) with high affinity but upon hCG stimulation in vitro, cAMP and testosterone production by these cells were minimal. On the other hand, the heavier cells displayed typical Leydig cell morphology and bound very little hCG but vigorously produced cAMP and testosterone (Browne, E.S., Bhalla, V.K., 1991, J. Androl. 12:132-139). This study examines the distribution of LH/hCG receptor mRNAs in the two cell types. The light cell fraction contains larger transcripts of LH/hCG receptor but the heavier Leydig cells contain shorter transcripts. The observations raises the intriguing possibility that shorter rather than larger LH/hCG receptor transcripts are responsible for the induction of a biologically functional, G-protein coupled, LH/hCG receptor in Leydig cells.


Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on insulin release from rat pancreatic islets. The role of protein phosphatases in the regulation of insulin release from rat pancreatic islets was studied with protein phosphatase inhibitors, okadaic acid and calyculin A. Okadaic acid inhibited glucose- and glyceraldehyde-induced insulin release dose-dependently and also inhibited the potentiation of glucose-induced release either by adding forskolin, an activator of adenylate cyclase or by increasing K+ concentration to 25 mM. At a non-stimulatory concentration of 3 mM glucose, a high concentration (2 microM) of okadaic acid inhibited insulin release induced by high K+ or 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, but a low concentration (1 microM) of okadaic acid did not significantly inhibit TPA-induced insulin release. Calyculin A also inhibited glucose-induced insulin release, and the effect was greater than that of okadaic acid. The data suggest that protein phosphatases may play an important role in the regulation of insulin release.


Preliminary evaluation of sonolaparoscopy in the diagnosis of liver diseases. A preliminary report is made on the use of sonolaparoscopy to overcome limitations of sonography and laparoscopy in the diagnosis of liver disease. The instruments used consisted of a sonolaparoscope with a mechanical radial scanner (LPSUMI/EUM1; Olympus) and the first prototype of an electronic sonolaparoscope (Olympus). Sonolaparoscopy was shown to be particularly suitable for detection and differentiation of occult liver lesions, cysts, tumors, various benign focal hyperplasias and various granulomata.


Treatment of infantile spasms: medical or surgical? Although infantile spasms were initially described in 1841, remarkably little progress has been made in understanding the pathophysiology of this "peculiar form of infantile convulsions." Consequently, our ability to treat infantile spasms is limited. Infantile spasms are classified as a "generalized" seizure disorder in the international classification system, which suggests that the underlying brain abnormality causing the seizures also must be diffuse or generalized. As the classification suggests, there are many diffuse, or multifocal, brain disorders related to infantile spasms, e.g., inborn errors of metabolism, hypoxic-ischemic brain injury, and developmental brain defects such as tuberous sclerosis or Aicardi's syndrome. On the other hand, infantile spasms have been reported in which a localized brain abnormality was present, e.g., tumor, stroke, and trauma. On rare occasions, removal of a tumor has resulted in cessation of the generalized infantile spasms. This finding suggests that focal cortical abnormalities can cause infantile spasms and that removing the abnormality can stop the seizures. At University of California, Los Angeles, the Pediatric Epilepsy Surgery Program has developed new approaches to the treatment of infantile spasms. The principal underlying concepts are (a) children with medically refractory infantile spasms may have an area of cortical defect (called the zone of cortical abnormality) that causes the seizures and (b) infantile spasms are usually generalized seizures. Thus, the goal of the surgical assessment is not the identification of the focus of seizure onset but rather the identification of the zone of cortical abnormality.


GABA and epilepsy: basic concepts from preclinical research. The role of gamma-aminobutyric acid (GABA) transmission in the control of convulsive epileptic seizures is considered from the perspective of the actions of drugs that augment GABA transmission in the brain. In particular, the effects of a directly acting GABAA receptor agonist, muscimol, is compared with the effects of a GABA-elevating agent, gamma-vinyl GABA (GVG, vigabatrin), in animal models of convulsive seizures. Evidence indicates that there are certain regions of the brain where enhanced GABA transmission is anticonvulsant; in other regions, blockade of GABA transmission exerts anticonvulsant actions. In addition, there are brain areas in which the effects of muscimol and GVG are distinct from one another, owing to a relatively low level of endogenous GABA transmission in those areas. The direct stimulation of postsynaptic GABA receptors (by direct receptor agonists) bypasses normal mechanisms of synaptic transmission and can evoke abnormal neurological symptoms, whereas the enhancement of presynaptic availability of GABA avoids these complications. GVG acts to boost presynaptic GABA stores, which can then be utilized physiologically; this may account for the relatively low incidence of CNS-related side effects with anticonvulsant doses of GVG. It is suggested that greater attention be focused on ways of enhancing endogenous GABA availability in future drug development for the control of seizure disorders.


Advances in lung cancer?

Advances in lung cancer? Selikoff was among the first to emphasize the etiologic role of cigarette smoking and asbestos exposure in lung cancer. Recent reports suggest that aggressive chemotherapy and radiotherapy combined with surgery have improved the prognosis in lung cancer. The outcome of 100 patients with non-small-cell lung cancer (NSCLC) seen in 1979-1980 was compared with the outcome of 100 patients seen in 1989-1990. The two groups consisted of consecutive patients with proven NSCLC who were referred to a pulmonary consultant. In 28 of the earlier patients and 38 of the later group the cancers were completely resectable. Only 12 of the 1979-1980 group have been cured of their cancer, while 25 of the 1989-1990 are free of cancer during the less than 2-year follow-up period. It can be assumed that this number will diminish with time. Current treatment of NSCLC with surgical resection, chemotherapy, and radiotherapy has done little to improve the prognosis of lung cancer in the past 10 years. As stressed by Selikoff, the best available current method for control of lung cancer is prevention.


Validity of repeated dietary measurements in a dietary intervention study. The aim of the study was to evaluate the compliance in a dietary intervention study. When drawing conclusions about the relationship between dietary intake and disease occurrence/disease-related variables it is important to obtain valid dietary data. 20 healthy, non-smoking normal-weight omnivores changed from a mixed to a lactovegetarian diet. Dietary surveys (four 24 h recalls per person and time-period), urinary and faecal sample collections were performed before and 3, 6 and 12 months after the dietary shift. The validation of energy, protein, sodium and potassium yielded approximately the same ratio of dietary intake to biological marker at 0 and 3 months. This ratio decreased towards 6 months and continued to decrease towards 12 months. The fibre intake was compared to the total faecal weight directly and indirectly by calculating the fibre intake from the stool weight, the water content in faeces and the excretion of short-chain fatty acids (SCFAs). These four methods of fibre validation showed that the ratio of dietary intake to biological marker was always highest at 12 months, indicating an overestimation of the fibre intake at the end of the study. This is the first time these methods of validating fibre intake have been used in an epidemiological study. The ratio of dietary calcium intake to urinary and faecal calcium excretion did not show any statistical difference between the period before and 3 months after the dietary shift. To conclude, almost all investigated dietary data show approximately the same validity before and 3 months after the dietary shift, and show the least validity 12 months after the dietary shift. Thus, this study demonstrates that it is difficult to obtain valid dietary data 1 year after a drastic dietary change, indicating a decreased compliance to the new dietary regimen at the end of the 1 year study period. This represents important information when attempting to relate biological effects to dietary intake, and illustrates the importance of using biological markers for food intake in dietary surveys.


Formation of resistant starch in deproteinized and non-deproteinized beans. Resistant starch (RS) was determined in deproteinized and non-deproteinized whole and milled beans (Phaseolus vulgaris) after cooking and freezing. The highest RS values were obtained in the whole bean preparations (3.7-8.7%). Milled and milled and deproteinized beans presented lower RS contents (1.64% and 0.91%, respectively). The results suggest that the presence of protein and the particle size are the main factors affecting the formation of RS.


Limiting factors of starch hydrolysis.

Limiting factors of starch hydrolysis. Foods appear as complex structures, in which starch may be present in different forms. These, including the molecular characteristics and the crystalline organization, depend on processing conditions and compositions of ingredients. The main changes in starch macro- and microstructures are the increase of surface area to volume ratio in the solid phase, the modification of the crystallinity as affected by gelatinization and gelation, and the depolymerization of amylose and amylopectin. Starch modification may be estimated by different methodologies, which should be selected according to the level of structure considered. When amylose and amylopectin are in solution, rapid and total hydrolysis leads to the formation of a mixture of linear oligosaccharides and branched alpha-limit dextrins. However, starch usually occurs in foods as solid structures. Structural factors of starchy materials influence their enzymic hydrolysis. A better understanding of the enzymatic process enables the identification of the structural factors limiting hydrolysis: diffusion of enzyme molecules, porosity of solid substrates, adsorption of enzymes onto solid substrates, and the catalytic event. A mechanistic modelling should be possible in the future.


Physical characteristics of starch granules and susceptibility to enzymatic degradation. Starch, the most abundant component of the diet, is characterized by its variety as well as the versatility of its derivatives in foods. This paper is an overview of the main physical characteristics of the native starch granule. Three different levels of organization are presented: macromolecular structure, crystalline organization and ultrastructure. Starch consists of amylose and amylopectin. Amylose is an essentially linear polymer composed of alpha-1,4-linked D-anhydroglucose units (AGU); amylopectin is a branched polymer clustering a large amount of short linear chains by the linkage of alpha-1,6-bonds, constituting about 5% of the total glycosidic bonds. In the native starch granules, a large number of the macromolecular chains are organized in crystalline structures. Three forms have been found, the A, B and C patterns. So far only A and B starch crystals have been modelled. There is a variation in the susceptibility of the starch granules to enzymatic digestion. This is explained by variation in the morphology of the granules and their crystalline organization.


Classification and measurement of nutritionally important starch fractions. For nutritional purposes, starch in foods may be classified into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). RS may be further divided into three categories according to the reason for resistance to digestion. A method is reported for the measurement of total starch, RDS, SDS, RS and three RS fractions in starchy foods, using controlled enzymic hydrolysis with pancreatin and amyloglucosidase. The released glucose is measured by colorimetry, using a glucose oxidase kit. Values for RDS and SDS in foods obtained by the method reflect the rate of starch digestion in vivo. Values for RS are similar to the amounts of starch escaping digestion in the small intestine of ileostomates, and are a guide to the amounts of starch likely to enter the colon for fermentation. Results are given for a number of starchy foods.


Determination of resistant starch in foods and food products: interlaboratory study. A collaborative study was conducted to compare methods of determining resistant starch (RS) in various foods and food products. Principally, two methods derived from Berry (1986, Journal of Cereal Science, 4, 301-314) (method A) and Björck et al. (1986, Journal of Cereal Science, 4, 1-11) (method B) were used to analyse four samples containing various forms of RS. Several methods were also used to determine total starch. The RS yield by method A compared to method B was higher in all samples except unmilled bean flakes. When the starch was a raw B-type (potato starch), method B failed to detect any RS because of its initial heating step, whereas method A yielded 48% RS on an 'as is' basis.


The ileostomy model for the study of carbohydrate digestion and carbohydrate effects on sterol excretion in man. The ileostomy model offers a method for direct, accurate and quantitative determination of small bowel excretion, provided the bacteriological degradation of the effluent can be minimized. The intraindividual variation is small, and rapid changes in excretion pattern due to dietary manipulations can be measured. Such ileostomy investigations have been important for developing the resistant starch and dietary fibre concepts. Using this method, various mechanisms for effects of dietary fibre on plasma lipids can be elucidated, e.g. binding of cholesterol, bile acids and/or lipids. Another application is to study the capacity of phytic acid and its degradation products to bind minerals in the small bowel.


Hydrogen and methane breath tests for evaluation of resistant carbohydrates. This review considers in detail the background, principles, techniques, limitations and advantages of the hydrogen and methane breath tests. Resistant food carbohydrates, defined as dietary carbohydrates partly or totally escaping small intestinal assimilation, are fermented in the human colon. This results in production of H2, CH4 and volatile fatty acids. Increased colonic H2 production is a sensitive index of increased carbohydrate fermentation, and a rather constant fraction of the colonic H2 production is excreted by the lungs. It is therefore possible to assess mouth-to-caecum transit times as well as to estimate absorption capacities for several types of resistant carbohydrates by means of H2 breath tests. A prerequisite for correct interpretation is that procedures for determination of H2 concentrations and for breath sampling and storage are carefully validated and standardized. Due to the large interindividual variations of hydrogen excretion, unabsorbable standards should be used. The intraindividual variations of H2 production/excretion and differences in fermentability of different carbohydrate substrates only allow for semiquantitative estimates of malabsorbed amounts of some carbohydrates. Methane breath tests may supplement the information gained from hydrogen measurements, but further evaluations are needed. The hydrogen breath technique is rapid, simple and non-invasive as well as non-radioactive. It may be carried out in a large number of intact individuals under physiological circumstances, and it may be used for studies in children and for field studies. Compared to classical tolerance tests the hydrogen breath test is more sensitive. It is concluded that the hydrogen breath test is a useful tool for investigations of dietary carbohydrates.


Glycaemic index of foods.

Glycaemic index of foods. From the mid-1970s several groups realized progressively that the same amounts of carbohydrates in different foods produce quite different blood glucose curves after ingestion. The glycaemic index (GI) was introduced by Jenkins to express the rise of blood glucose after eating a food against a standard blood glucose curve after glucose (or white bread) in the same subject. The GI ranges from about 20 for fructose and whole barley to about 100 for glucose and baked potato. A table is given of representative GI values. There appears to be no general correlation between GI and per cent resistant starch in foods. Questions about methodology for GI are discussed and the factors in food that affect glycaemic response are briefly reviewed. The GI is affected by the physical form of a food, by processing and by associated fat in the food, which reduces the GI, presumably by delayed gastric emptying. As a rule the degree of insulin response to carbohydrate-containing foods is similar to the glycaemic response. Most investigators have found that the GI of a meal of mixed foods can be predicted from the (weighted) GI of its constituent foods. The GI concept is proving useful in dietary design for the management of diabetes mellitus, especially the non-insulin-dependent type. It may prove useful for prevention of diabetes and perhaps also in pre-event meals for athletes, as a factor in dental cariogenesis, in determining satiety, and conceivably regular low GI foods could delay ageing by reducing glycosylation of body proteins.


Distinction between supercoiled and linear DNA in transverse agarose pore gradient gel electrophoresis. Four species of linear DNA and the first four members of a linking series, generated by treatment of plasmid DNA (PUC19, 2.7 kb) with mitochondrial topoisomerase I, were differentiated by transverse agarose pore gradient gel electrophoresis. The experimental curves of migration distance vs. agarose concentration (Ferguson curves) of supercoiled DNA exhibit a steeper trajectory than those of linear DNA of the same size range. As a consequence, the four supercoiled species exhibit an increase in apparent size (relative to linear DNA standards) with increasing agarose concentration. Both the crossing of the Ferguson curves with those of linear standards as well as the apparent size increase with agarose concentration can serve to detect supercoiled plasmid-sized DNA in mixtures with linear DNA.


Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes. Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed.


Isolation and cloning of Omp alpha, a coiled-coil protein spanning the periplasmic space of the ancestral eubacterium Thermotoga maritima. We have discovered a new oligomeric protein component associated with the outer membrane of the ancestral eubacterium Thermotoga maritima. In electron micrographs, the protein, Omp alpha, appears as a rod-shaped spacer that spans the periplasm, connecting the outer membrane to the inner cell body. Purification, biochemical characterization and sequencing of Omp alpha suggest that it is a homodimer composed of two subunits of 380 amino acids with a calculated M(r) of 43,000 and a pI of 4.54. The sequence of the omp alpha gene indicates a tripartite organization of the protein with a globular NH2-terminal domain of 64 residues followed by a putative coiled-coil segment of 300 residues and a COOH-terminal, membrane-spanning segment. The predicted length of the coiled-coil segment (45 nm) correlates closely with the spacing between the inner and outer membranes. Despite sequence similarity to a large number of coiled-coil proteins and high scores in a coiled-coil prediction algorithm, the sequence of the central rod-shaped domain of Omp alpha does not have the typical 3.5 periodicity of coiled-coil proteins but rather has a periodicity of 3.58 residues. Such a periodicity was also found in the central domain of staphylococcal M protein and beta-giardin and might be indicative of a subclass of fibrous proteins with packing interactions that are distinct from the ones seen in other two-stranded coiled-coils.


Native mRNA editing complexes from Trypanosoma brucei mitochondria. The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10-30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre-edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA-mRNA chimera-forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera-forming activity. Complex II (35-40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera-forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.


Frequent transpositions of Drosophila melanogaster HeT-A transposable elements to receding chromosome ends. HeT-A elements are a new family of transposable elements in Drosophila that are found exclusively in telomeric regions and in the pericentric heterochromatin. Transposition of these elements onto broken chromosome ends has been implicated in chromosome healing. To monitor the fate of HeT-A elements that had attached to broken ends of the X chromosome, we examined individual X chromosomes from a defined population over a period of 17 generations. The ends of the X chromosomes with new HeT-A additions receded at the same rate as the broken ends before the HeT-A elements attached. In addition, some chromosomes, approximately 1% per generation, had acquired new HeT-A sequences of an average of 6 kb at their ends with oligo(A) tails at the junctions. Thus, the rate of addition of new material per generation matches the observed rate of terminal loss (70-75 bp) caused by incomplete replication at the end of the DNA molecule. One such recently transposed HeT-A element which is at least 12 kb in length has been examined in detail. It contains a single open reading frame of 2.8 kb which codes for a gag-like protein.


A regulatory element that mediates co-operation between a PEA3-AP-1 element and an AP-1 site is required for phorbol ester induction of urokinase enhancer activity in HepG2 hepatoma cells. We have characterized a transcriptional enhancer of the human urokinase-type plasminogen activator (uPA) gene and found a regulatory element required for co-operation between a PEA3--AP-1 element and an AP-1 site in the enhancer. We designated this regulatory element co-operation mediator (COM). Both the PEA3--AP-1 element, the AP-1 site and the COM are required for efficient phorbol ester induction of transcription from the uPA promoter in the HepG2 hepatoma cell line. We show that the COM is also required for co-operation between the PEA3--AP-1 element and a glucocorticoid response element, both in the presence or absence of TPA, indicating that the COM is generally capable of mediating synergism between inducible enhancer elements. The COM contains multiple overlapping binding sites for nuclear proteins, designated uPA enhancer factors 1-4 (UEF-1-4). We have identified putative binding sites for UEF-1, -2 and -3. The UEF-1 and -3 sites in the uPA enhancer are highly conserved between species. We demonstrate the binding of UEF-3 to the NIP element, a previously characterized regulatory element in the human interleukin-3 and stromelysin promoters, suggesting that this factor plays a role in regulation of a variety of genes.


The 55 kDa regulatory subunit of protein phosphatase 2A plays a role in the activation of the HPV16 long control region in human cells with a deletion in the short arm of chromosome 11. Previous results indicated that SV40 small t is essential for SV40-induced transformation of diploid cells but dispensable for the transformation of cells with a deletion on the short arm of chromosome 11 (del-11 cells). From these results we concluded that del-11 cells contain a cellular 'SV40 small t-like' factor, which is able to transactivate the HPV16 long control region (LCR) and to complement SV40 large T in transformation. Since SV40 small t and the regulatory 55 kDa subunit (PR55) of protein phosphatase 2A (PP2A), have been shown to inhibit the enzyme activity of PP2A, the PR55 beta subunit could be the putative 'small t-like' factor. In accordance with this hypothesis, we show that the PR55 beta subunit is highly expressed in del-11 but not in diploid cells and is able to trans-activate the HPV16 LCR in diploid cells. Moreover, inhibition of PP2A by okadaic acid resulted in trans-activation of the HPV16 LCR in diploid cells. Alignment of PR55 and SV40 small t showed a common four amino acid motif DKGG. We present evidence that the integrity of this motif is necessary for the PP2A-mediated ability of SV40 small t to trans-activate the HPV16 LCR.


Identification of a negative regulatory domain in the human papillomavirus type 18 promoter: interaction with the transcriptional repressor YY1. The human papillomavirus type 18 (HPV-18) promoter contains a TPA responsive element (TRE) which confers TPA responsiveness on a heterologous promoter. In the context of the HPV-18 promoter, however, this AP-1 site is inactive. We have identified a negative regulatory domain in the HPV-18 promoter which represses the constitutive and TPA-induced AP-1 activity. This negative regulatory sequence has been mapped to 44 nucleotides (OL13). We identified this element as a transcriptional silencer based on its ability to interfere with transcriptional initiation. This HPV-18 silencer domain was narrowed down further to 23 nucleotides, the OL13B element, which bears similarity to three other silencer sequences, present in the mouse N-ras gene upstream regulatory region, the mouse albumin gene enhancer and the adeno-associated virus P5 promoter. The transcriptional repressor protein YY1, which negatively regulates the P5 promoter, binds to the HPV-18 silencer with high affinity. Mutation of the YY1 binding site leads to an enhanced activity of the HPV-18 promoter, strongly suggesting that YY1 plays an important role in controlling HPV-18 early gene expression.


Transcription of human c-myc in permeabilized nuclei is associated with formation of Z-DNA in three discrete regions of the gene. When human U937 cells are placed in agarose microbeads and treated with a detergent, the cytoplasmic membrane is lysed and the nuclear membrane is permeabilized. However, the nuclei remain intact and maintain both replication and transcription. Biotin labeled monoclonal antibodies against Z-DNA have been diffused into this system and used to measure the amount of Z-DNA present in the nuclei. It has previously been shown that the amount of Z-DNA present decreases due to relaxation by topoisomerase I and increases as the level of transcription increases. Here we measure the formation of Z-DNA in the c-myc gene by crosslinking the antibodies to DNA using laser radiation at 266 nm for 10 ns. The crosslinked DNA is isolated by restriction digestion, separation of antibody labeled fractions through the biotin residue, and subsequent proteolysis to remove the crosslinked antibody. Three AluI restriction fragments of the c-myc gene are shown to form Z-DNA when the cell is transcribing c-myc. The Z-DNA forming segments are near the promoter regions of the gene. However, when U937 cells start to differentiate and transcription of the c-myc gene is down-regulated, the Z-DNA content goes to undetectable levels within 30-60 min.


Investigation of the mutagenic specificity of X-rays using a retroviral shuttle vector in CHO cells. In some biological systems ionizing radiation appears to induce large deletions and rearrangements, while in others point mutations predominate the mutational spectrum. Moreover, while the point mutations are often randomly distributed, some systems exhibit "hot spots." Retroviral shuttle vectors are particularly useful for investigating the basis of these differences since the genetic target can be conveniently analyzed in a variety of host backgrounds and genomic locations. We have studied the mutational specificity of X-rays in a Chinese hamster ovary cell line (CHO) containing a stably integrated retroviral shuttle vector, carrying the CHO aprt cDNA as the genetic target. Cells were irradiated with 7 Gy using a 180 kVp X-ray source. The predominant mutation (87% of all APRT mutants), as determined by Southern analysis, was the complete deletion of the shuttle vector construct. In addition, 23 APRT mutants, carrying an apparently intact shuttle vector, were characterized at the sequence level: 5 were transitions, 9 were transversions, 3 were small deletions or insertions, 4 were frameshifts, and 2 were small rearrangements. Although the type and the location of the point mutations characterized appeared largely random, small deletions, insertions, and frameshifts were frequently associated with direct sequence repeats.


Analysis of DNA strand breaks induced in rodent liver in vivo, hepatocytes in primary culture, and a human cell line by chlorinated acetic acids and chlorinated acetaldehydes. An alkaline unwinding assay was used to quantitate the induction of DNA strand breaks (DNA SB) in the livers of rats and mice treated in vivo, in rodent hepatocytes in primary culture, and in CCRF-CEM cells, a human lymphoblastic leukemia cell line, following treatment with tri- (TCA), di- (DCA), and mono- (MCA) chloroacetic acid and their corresponding aldehydes, tri- (chloral hydrate, CH), di- (DCAA) and mono- (CAA) chloroacetaldehyde. None of the chloroacetic acids induced DNA SB in the livers of rats at 4 hr following a single administration of 1-10 mmole/kg. TCA (10 mmole/kg) and DCA (5 and 10 mmole/kg) did produce a small amount of strand breakage in mice (7% at 4 hr) but not at 1 hr. N-nitrosodiethylamine (DENA), an established alkylating agent and a rodent hepatocarcinogen, produced DNA SB in the livers of both species. TCA, DCA, and MCA also failed to induce DNA strand breaks in splenocytes and epithelial cells derived from the stomach and duodenum of mice treated in vivo. None of the three chloroacetaldehydes induced DNA SB in either mouse or rat liver. The continuous exposure of mice to 5 g/L DCA in the drinking water for 7 and 14 days did not induce appreciable hepatic DNA SB (< 10% at 14 days), although peroxisome proliferation, as evidenced by an increased cyanide-insensitive palmitoyl CoA oxidase (PCO) activity, was stimulated to 490% (7 days) and 652% (14 days) of control. Under this protocol, DENA (0.1 g/L) produced DNA damage after both 7 days (73% of control) and 14 days (57% of control). Similarly, long-term exposure of rats (30 weeks) to 2 g/L DCA in the drinking water, a level that increased PCO activity to 364% of the control value, exhibited no DNA damage. Both the chloroacetic acids and the chloroacetaldehydes were ineffective in inducing DNA SB in cultured rat and mouse hepatocytes at concentrations below those that yielded cytotoxicity. The chloroacetic acids were also ineffective in the CCRF-CEM cells. However, two of the chloroaldehydes, DCAA and CAA, did induce DNA SB in the CCRF-CEM cells at concentrations that did not decrease the cell viability after 2 hr of treatment. Prior incubation of DCAA and CAA with a rat S9 liver homogenate eliminated much of the DNA damaging activity. These studies provide further evidence that the chloroacetic acids lack genotoxic activity not only in rodent liver, a tissue in that they induce tumors, but in a variety of other roden tissues and cultured cell types.(ABSTRACT TRUNCATED AT 400 WORDS)


Certain tryptophan photoproducts are inhibitors of cytochrome P450-dependent mutagenicity. Two photoproducts, derived from UV-irradiation of the amino acid L-tryptophan and with high Ah (TCDD) receptor binding affinity, were tested for genotoxic and antimutagenic effects. The two indolo[3,2-b]carbazole derivatives, with the molecular weights of 284 and 312, respectively, were tested in Saccharomyces cerevisiae strain D7 for mitotic gene conversion and reverse mutation and in strain RS112 for sister chromatid conversion and gene conversion. No significant (P > 0.05) genotoxic effects were found in strain D7, while strain RS112 showed a small but significant increase in the frequency of sister chromatid conversions. In Chinese hamster ovary (CHO) cells the two compounds induced a statistically significant but less than twofold increase in the frequency of sister chromatid exchanges (SCE). No mutations were detected when the compounds were tested in Salmonella typhimurium strains TA98 and TA100. However, both 284 and 312 acted as antimutagens on strain TA100 + S9 in the presence of benzo(a)pyrene. The decrease in mutagenicity by the most potent compound 284 was 20 revertants/nmol. This effect could be explained by an inhibitory effect on the cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity as seen in rat hepatocytes. The two compounds were also tested with hamster cells expressing rat cytochrome P-450IA1. The results support the conclusion that this cytochrome P-450 isozyme is inhibited by the tryptophan photoproducts. Similar results were also seen with two other high affinity Ah receptor ligands the quinazolinocarboline alkaloids rutaecarpine and dehydrorutaecarpine.


The delta receptor is involved in sufentanil-induced respiratory depression--opioid subreceptors mediate different effects. It is generally accepted that analgesia induced by central analgesics is mediated through the mu-receptor. However, it still remains open to question as to whether or not the mu- and/or the delta-receptor site is mainly involved in the mediation of opioid-related respiratory impairment. Using a highly selective antagonist, naltrindole (NTI), or its benzofuran analogue naltriben (NTB), the hypothesis that competitive antagonism at the delta-receptor is able to attenuate sufentanil-related respiratory depression was tested in the dog. High dose (20 micrograms kg-1) sufentanil-induced respiratory impairment could be reversed by selective NTI-antagonism in a dose-related fashion (40-80-160 micrograms kg-1) increasing PaO2 from 57 to 81 mmHg and lowering PaCO2 from 52.1 to 49.2 mmHg. NTB-antagonism (40-80-160 micrograms kg-1) increased PaO2 from 48.4 to 91.2 mmHg and reduced PaCO2 from 46.9 to 37.6 mmHg. Simultaneously, somatosensory-evoked potentials (SEP) were used to quantify the opioid-induced attenuation and the reversal of afferent sensory input to pain modulating centres in the CNS. Sufentanil induced a significant depression (P < 0.01) of amplitude height of the SEP (13.9 to 0.9 microV in the NTI- and 8.8 microV to 1.3 microV in the NTB-group) which was only partially reversed by NTI (2.6 microV) and NTB (2.3 microV) respectively. The results suggest that delta-receptors are involved in sufentanil-related respiratory impairment. These receptors play a minor role in opioid-induced attenuation of sensory input to the brain. Highly selective delta-antagonists may be of clinical interest in reversing the respiratory depressant effect of potent opioids while maintaining analgesia.


Plasma potassium and ventilation during incremental exercise in humans: modulation by sodium bicarbonate and substrate availability. It has recently been demonstrated that, compared to normal conditions, ventilation (VE) was increased during exercise after glycogen depletion, in spite of a marked increase in plasma pH (pHP). It was further demonstrated that VE in patients with McArdle's syndrome was reduced when substrate availability was improved. In the present experiments, six endurance trained men performed two successive cyclo-ergometric incremental exercise tests (tests A, B) after normal nutrition (N) and after a fatty meal in conjunction with a sodium bicarbonate (NaHCO3) solution (FSB) or without NaHCO3 (F), and the relationship between VE, plasma potassium concentration ([K+]P), and pHP was checked. Plasma free fatty acid concentration ([FFA]P) was markedly increased in the F and FSB trials (P < 0.001). In FSB pHP was significantly increased, compared to N and F (P < 0.001). In all the B tests, pHP increased during moderate and intense exercise and in FSB, remained alkalotic even during maximal exercise intensity. In contrast, VE and [K+]P changes were almost equal in all the trials and in tests A and B. It was found that exercise-induced changes of VE and [K+]P in the present experiments were not markedly affected by [FFA]P or pHP values and that these changes also occurred independently of changes in pHP or plasma bicarbonate concentration. The often used glycogen depletion strategy may have slightly increased VE but apparently did not overcompensate for a possible decrease in VE due to increased pHP.(ABSTRACT TRUNCATED AT 250 WORDS)


The elusive nuclear matrix.

The elusive nuclear matrix. The structure of the interphase nucleus is a major area of current interest in cell biology. It is thought likely that the nucleus is organised around some form of structural matrix and that this matrix will play a role in processes as diverse as chromosome replication and the integration of gene expression. However, the structure of the matrix within the nucleus has remained elusive, largely because attempts to define it have been dogged by technical problems arising from the great complexity of this organelle. This situation is now being changed by the application of in situ analysis and of molecular genetic methodologies which are opening up this hitherto intractable field.


X-ray crystallographic characterisation of type-2-depleted ascorbate oxidase from zucchini. The type-2 depleted form of ascorbate oxidase from zucchini has been prepared in crystals and characterised by X-ray crystallography and EPR spectroscopy. The X-ray structure analysis by difference-Fourier techniques and refinement shows that, on average, about 1.3 Cu atoms/ascorbate oxidase monomer are removed. The copper is lost from the trinuclear site whereby the EPR-active type-2 copper is depleted most; type-1 copper is not affected. This observation indicates preferential formation of a 1 Cu-depleted form with the hole equally distributed over all three copper sites. Each of these 1 Cu-depleted species may represent an anti-ferromagnetically coupled copper pair which is EPR-silent and could explain the disappearance of the type-2 EPR signal.


Ferrisiderophore reductases of Pseudomonas. Purification, properties and cellular location of the Pseudomonas aeruginosa ferripyoverdine reductase. Purification of the ferripyoverdine reductase from Pseudomonas aeruginosa, strain PAO1, lead to the isolation of a soluble protein of M(r) 27,000-28,000, as determined by HPLC sieving filtration and by denaturating gel electrophoresis. In the presence of NADH as the reductant, ferripyoverdine as the iron substrate, ferrozine as an iron(II)-trapping agent and FMN, this protein displayed an iron-reductase activity which resulted in the formation of ferrozine-iron(II) complex, providing that the enzymic assay was run under strict anaerobiosis. FMN was absolutely required for the activity to occur, but the lack of a visible spectrum and the lack of fluorescence for the protein in solution suggested that ferripyoverdine reductase is not a flavin-containing protein and that covalently bound FMN is not a prerequisite for the enzymatic reaction. A search of ferripyoverdine reductase by immunological detection amongst the different cellular compartments of P. aeruginosa lead to the conclusion that the soluble enzyme, which represented more than 95% of the total cellular enzyme, is not located in the periplasm but specifically in the cytoplasm. A strongly immunoreacting material, corresponding to a protein with identical M(r) as the ferripyoverdine reductase of P. aeruginosa PAO1, was detected in all the eighteen fluorescent pseudomonad strains belonging to the P. aeruginosa, P. fluorescens, P. putida and P. chlororaphis species, as well as in P. stutzeri, a non-fluorescent species, suggesting that the enzyme acting as a ferripyoverdine reductase in P. aeruginosa PAO1 is ubiquitous among the Pseudomonas.


Biochemical characterization of thioredoxin 1 from Dictyostelium discoideum. Multiple genes for thioredoxins (TRX) have been demonstrated in Dictyostelium discoideum. We expressed the cDNA for one of these genes (DdTrx1) in E. coli and purified the protein to homogeneity. The interaction with classic substrates as well as TRX reductases was analysed. It reacted with every tested substrate: insulin, NADP-dependent malate dehydrogenase and fructose-1,6-bisphosphatase. With a S0.5 of 20 microM, the reactivity with the fructose-1,6-bisphosphatase is the highest ever found for a heterologous TRX. DdTRX1 itself is accepted as a substrate by the chloroplast ferredoxin-dependent TRX reductase, as well as by the E. coli NADPH-dependent TRX reductase. Thus, the Dictyostelium TRX is functionally promiscuous. Its reactivity with insulin, chloroplast NADP-dependent malate dehydrogenase and ferredoxin-dependent TRX reductase resemble those of other TRX. It is, however, clearly different in its good interaction with chloroplast fructose-1,6-bisphosphatase and in its poor interaction with E. coli NADP-dependent TRX reductase.


Characterization of Saccharomyces cerevisiae mutants lacking the E1 alpha subunit of the pyruvate dehydrogenase complex. Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDA1 gene. To this end, the PDA1 gene encoding the E1 alpha subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker. Disruption of the PDA1 gene abolished production of the E1 alpha subunit and pyruvate dehydrogenase activity. Two additional phenotypes were observed in the Pdh-mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA [rho0], during growth on glucose. Both phenotypes were shown to be a result of inactivation of the PDA1 gene. Explanations for these phenotypes are discussed.


Beta-adrenergic stimulation of C6 glioma cells: effects of cAMP overproduction on cellular metabolites. A multinuclear NMR study. We used 31P-NMR spectroscopy to investigate the response of living C6 glioma cells to stimulation by a beta-adrenergic agonist, isoproterenol. In the presence of 3-isobutyl-1-methylxanthine, stimulation induced an accumulation of cAMP, making possible the NMR detection of the second messenger in living cells grown on microcarrier beads and perfused in the NMR tube. The cAMP signal rose to a maximum level within 20-25 min of stimulation; thereafter it decreased to the detection threshold within 60 min. At the same time, 40% increases of phosphomonoester and diphosphodiester signals were observed, whereas no significant change in phosphocreatine and nucleotide signals was detected. The kinetics of changes of the cellular content in phosphorylated metabolites were analyzed after recording 31P-NMR spectra of cell perchloric acid extracts as a function of time of stimulation. cAMP accumulation in stimulated cells was evidenced by a near linear increase of its NMR signal as a function of incubation time (from 0 to 60 min). Concomitantly with the production of cAMP, the data showed 30% decreases of phosphocreatine and ATP levels within 60 min of stimulation, and an unexpected redistribution of pyrimidine and purine nucleoside triphosphates. At the same time, levels of phosphomonoesters (phosphorylcholine and phosphorylethanolamine) and phosphodiesters (glycerophosphorylcholine and glycerophosphorylethanolamine) rose (50% increase). 13C-NMR spectra of cell perchloric acid extracts prepared after isoproterenol stimulation of cells incubated in the presence of [1-13C]glucose indicated a higher glucose content in stimulated cells, whereas the resonance of ribose C1 was diminished. Moreover, the resonances of C1 of ethanolamine and choline (and their derivatives) were increased in spectra of stimulated cells, whereas that of C3 of serine was decreased. In addition, the 13C-NMR data indicated that neither the pattern of glutamate carbon enrichment nor the glutamate/glutamine ratio was modified in stimulated cells. On the other hand, the heteronuclear coupling pattern of the lactate (methyl group) resonance in 1H-NMR spectra of cell incubation media indicated that no change occurred in the carbon flux through the pentose-phosphate shunt under stimulation. The results of this multinuclear NMR approach are discussed in terms of metabolic responses of C6 cells to beta-adrenergic stimulation and cAMP overproduction.


Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus. The enzyme carboxyphosphonoenolpyruvate mutase catalyses the formation of one of the two C-P bonds in bialaphos, a potent herbicide isolated from Streptomyces hygroscopicus. The gene encoding the enzyme has been cloned from a subgenomic library from S. hygroscopicus by colony hybridisation using an exact nucleotide probe. An open reading frame has been identified that encodes a protein of molecular mass 32700 Da, in good agreement with the subunit molecular mass of the carboxyphosphonoenolpyruvate mutase recently isolated from this source [Hidaka, T., Imai, S., Hara, O., Anzai, H., Murakami, T., Nagaoka, K. & Seto, H. (1990) J. Bacteriol. 172, 3066-3072]. The gene shares significant sequence similarity with that of phosphoenolpyruvate mutase, an enzyme that catalyses the related interconversion of phosphoenolpyruvate and phosphonopyruvate. When the carboxyphosphonoenolpyruvate-mutase gene was subcloned into the vector pET11a, the mutase was expressed as about 20% of the total soluble cellular protein in Escherichia coli. The mutase has been purified to homogeneity in three steps in 40% yield. With malate dehydrogenase/NADH, (hydroxyphosphinyl)pyruvate gives (hydroxyphosphinyl)lactate (kcat 164 s-1 and Km 680 microM) and this spectrophotometric assay for the product of the mutase reaction has been employed in the mechanistic studies. The kinetics for the mutase reaction have been evaluated for the substrate, carboxyphosphonoenolpyruvate, and for the putative reaction intermediate carboxyphosphinopyruvate, both of which have been prepared by chemical synthesis. Carboxyphosphonoenolpyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 0.020 s-1 and a Km of 270 microM, and carboxyphosphinopyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 7.6 x 10(-4) s-1 and a Km of 2.2 microM. Although the exogenously added intermediate is not kinetically competent, these results suggest that the mechanism for the mutase reaction involves an initial rearrangement to the intermediate carboxyphosphinopyruvate, followed by decarboxylation to yield the product (hydroxyphosphinyl)pyruvate.


A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei. We have recently reported that the thermophilic archaeon Methanobacterium wolfei contains two formylmethanofuran dehydrogenases, I and II. Formylmethanofuran dehydrogenase II, which is preferentially expressed in tungsten-grown cells, has been purified and shown to be a tungsten-iron-sulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum-grown cells and shown that it is a molybdenum-iron-sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per mol 146-kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non-heme iron but no tungsten (< 0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 U/mg) in that it catalyzed the oxidation of N-furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent Km for the electron acceptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N-terminal amino acid sequences determined for the alpha and beta subunits were identical up to residue 23, indicating that the two proteins are isoenzymes. The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.


On the stoichiometry of the iron-sulphur clusters in mitochondrial NADH: ubiquinone oxidoreductase. The concentration of the iron-sulphur (Fe-S) cluster 1b, present in complex I or soluble high-molecular-mass NADH dehydrogenase, was determined using different methods. It was found that direct double integration of the EPR signal at temperatures higher than 40 K, as is commonly used in this field of research, results in a considerable overestimation of the concentration of cluster 1b. It is demonstrated that this is caused by contributions from the relaxation-broadened signals of the Fe-S clusters 2-4 in the enzyme. The correct way for determining the intensity of the EPR signal of cluster 1b is by comparison with a simulated line shape. It is concluded that the concentration of cluster 1b is half that of cluster 2. This corroborates our proposal based on presteady-state kinetic and inhibitor-titration studies [Van Belzen, R., Van Gaalen, M. C. M., Cuypers, P. A. & Albracht S. P. J. (1990) Biochim. Biophys Acta 1017, 152-159] that the minimal functional unit of mitochondrial NADH:ubiquinone oxidoreductase must be a heterodimer.


A comparative EPR investigation of the multicopper proteins nitrous-oxide reductase and cytochrome c oxidase. The multicopper proteins, nitrous-oxide reductase (N2OR) and cytochrome c oxidase (COX), were investigated by EPR spectroscopy at microwave frequencies 2.4-35 GHz. Our results support a Cu-Cu interaction in COX and N2OR. At least 10 lines in the 2.7-GHz, 12 lines in the 4.6-GHz and 14 lines in the 9.2 GHz spectra were resolved for N2OR. Eight copper lines at 2.7 GHz, about nine lines at 4.6 GHz and about six lines at 9.2 GHz were resolved for COX. Simulations of the EPR spectra were consistent with most of the resonances of the multiline spectra, including regions in the center of the spectra where overlap of the three seven-line patterns is proposed. These simulations indicated that Cu-Cu interaction, in a mixed-valence [Cu(1.5) ... Cu(1.5)], S = 1/2 site is consistent with, if not proof of, the unusual spectral features observed for N2OR and COX.


Prevalence of genital human papillomavirus infections in patients at a sexually transmitted diseases clinic. The human papillomavirus was detected in cervicovaginal cells by the polymerase chain reaction in 14 of 37 (37.8%) patients attending a sexually transmitted disease (STD) clinic and in 6 of 43 healthy young women (14.0%) undergoing routine gynecologic examinations who served as controls. The results indicated that even the more malignant types of human papillomaviruses were not uncommon among the control group, and that the prevalence of human papillomavirus infection was significantly higher in STD clinic patients than in the control group. These findings confirm the suggestion that factors other than human papillomavirus infections may be involved in the pathogenesis of cervical cancer.


In vitro activity of the new glycopeptide decaplanin. The activity of decaplanin, a new glycopeptide, was compared to that of vancomycin, teicoplanin and daptomycin. Decaplanin was two- to four-fold less active than vancomycin, telcoplanin and daptomycin against Staphylococcus aureus and Staphylococcus epidermidis, with an MIC90 of 2 micrograms/ml for methicillin-susceptible and 4 micrograms/ml for methicillin-resistant isolates. Decaplanin had activity similar to that of vancomycin against Streptococcus pyogenes, Streptococcus agalactiae, group C and G streptococci, with an MIC90 of 0.12 micrograms/ml. It was less active than the other agents against the viridans group streptococci (MIC90 4 micrograms/ml). The activity of decaplanin against enterococci (MIC90 4 micrograms/ml) was similar to that of vancomycin. Clostridium spp. were inhibited by 0.5 micrograms/ml, peptostreptococci and peptococci by 0.25 microgram/ml. Decaplanin was active from pH 5.5 to 7.5. Inoculum size had a minimal effect on MICs, and increased concentrations of Ca2+ and Mg2+ and 50% serum did not alter MICs or MBCs.


Interpretive criteria for CI-960, fleroxacin and temafloxacin susceptibility tests with Haemophilus influenzae. Haemophilus influenzae strains with varied ampicillin resistance and beta-lactamase production patterns were tested against three investigational fluorinated quinolones (CI-960, fleroxacin, temafloxacin) using Haemophilus Test Medium (HTM) and National Committee for Clinical Laboratory Standards (NCCLS) methods. The disk diffusion zones and MICs were compared and regression statistics and scattergrams generated. The rank order of the agents according to activity against Haemophilus influenzae was CI-960 (MIC50 0.002 microgram/ml) greater than temafloxacin (MIC50 0.015 microgram/ml) greater than fleroxacin (MIC50 0.03 microgram/ml). The recommended susceptibility interpretive criteria for the 5-micrograms disks of each drug were: for CI-960 greater than or equal to 23 mm (MIC correlate less than or equal to 1 microgram/ml); for fleroxacin greater than or equal to 19 mm (MIC correlate less than or equal to 2 micrograms/ml); and for temafloxacin greater than or equal to 16 mm (MIC correlate less than or equal to 2 micrograms/ml). All recent Haemophilus influenzae isolates tested were susceptible to these potent fluoroquinolones and no interpretive errors were observed.


Inhibition of motility of Pseudomonas aeruginosa and Proteus mirabilis by subinhibitory concentrations of azithromycin. Exposure to subinhibitory concentrations (4-8 micrograms/ml) of azithromycin resulted in loss of motility in Proteus mirabilis strains and a significant reduction of motility in Pseudomonas aeruginosa strains. Examination revealed that the loss of motility was due to a total absence of flagella in Proteus mirabilis while the poor motility observed in Pseudomonas aeruginosa was due to absence of flagella in the majority of the population. Since motility may be considered a pathogenicity trait in the two species, these results confirm the unusual ability of azithromycin to reduce the expression of virulence factors in gram-negative pathogens.


Efficacy of single-dose azithromycin versus doxycycline in the treatment of cervical infections caused by Chlamydia trachomatis. The efficacy of single-dose azithromycin therapy in the treatment of cervical Chlamydia trachomatis infections was compared to that of a standard seven-day course of treatment with doxycycline. Cervical samples from 60 patients reacted positively in an enzyme immunoassay for detection of Chlamydia trachomatis. In 31 patients Chlamydia trachomatis was isolated from the sample taken before treatment. Fourteen of the 31 patients were treated with doxycycline and 17 with azithromycin. All cultures of samples taken one and four weeks after the start of therapy were negative. All 31 isolates showed a similar pattern of MICs for the seven antibiotics tested, including azithromycin and doxycycline. No differences were observed between isolates of different serovars. In samples from four patients chlamydial DNA could be detected by PCR one week after the start of the therapy and in two patients also after four weeks. No difference in microbiological parameters could be observed between the two treatment groups. It is concluded that single-dose azithromycin is as effective as a seven-day course of doxycycline in the therapy of cervical Chlamydia trachomatis infections.


Comparison of fixed concentration and fixed ratio options for testing susceptibility of gram-negative bacilli to piperacillin and piperacillin/tazobactam. Piperacillin combined with tazobactam was tested at both a fixed ratio (8:1) and fixed tazobactam concentration (4 micrograms/ml) against 2,685 consecutively isolated gram-negative bacilli and 56 highly piperacillin-resistant isolates. Tazobactam significantly enhanced the spectrum of piperacillin activity. Overall, at a concentration of 16 micrograms/ml piperacillin alone inhibited 78.8% of the Enterobacteriaceae isolates compared to inhibition of 92.7% and 95.5% by the 8:1 ratio and fixed (4 micrograms/ml) tazobactam combinations, respectively. In MIC tests the two combination options performed comparably against both routine and highly piperacillin-resistant isolates. Synergistic inhibition was observed for comparable numbers of isolates with the two combination options, the most marked effect being seen in the more highly piperacillin-resistant isolates. Both testing options are supported by the available human pharmacokinetic data; however the 8:1 ratio of piperacillin to tazobactam may be preferable given that the clinical formulation contains the two compounds in an 8:1 ratio and this ratio is maintained in vivo.


Isolation and partial characterization of the luminal plasmalemma of microvascular endothelium from rat lungs. This paper describes a procedure for isolating in high yield and at a high degree of purity the endothelial luminal plasmalemma from the microvasculature of the rat lung. The procedure relies on the modification of the density of the luminal plasmalemma obtained by coating it by perfusion in situ first, with cationized colloidal silica and then with Na polyacrylate. These steps generate a strongly adhering coat to the luminal plasmalemma that resists tissue homogenization to yield, upon repeated centrifugation through Nycodenz density gradients, a nearly homogeneous fraction of coated luminal plasmalemmal fragments still carrying their associated plasmalemmal vesicles. The fraction is enriched in the luminal plasmalemmal antigen, angiotensin converting enzyme, contains gp60, an antigen expected to occur on both plasmalemmal domains, is not enriched in either alkaline phosphatase or 5'-nucleotidase activity and is free of the mitochondrial and endoplasmic reticulum antigens so far tested. This procedure, that can be extended--in principle--to any vascular bed, obviates the use of cultured cells for studying the biochemistry of the endothelium, at least as far as the luminal endothelial plasmalemma is concerned.