Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Perturbation data”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 685 records · Page 38Linked to original sources
PubMed1988

Noncoordinate regulation of cardiac Gs protein and beta-adrenergic receptors by a physiological stimulus, chronic dynamic exercise.

We used a physiological stimulus, chronic dynamic exercise, in pigs to examine resultant changes in chronotropic responsiveness to catecholamine and biochemical features of cardiac beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein, GS. Long-term treadmill running resulted in a substantial (44%) down-regulation of right atrial beta-adrenergic receptors, but the dose of isoproterenol yielding a 50% maximal increase in heart rate was decreased by 57% (from 0.07 +/- 0.03 to 0.03 +/- 0.01 microgram/kg; P less than 0.02) despite this decrease in receptor number. This disparity between receptor number and physiological responsiveness suggested altered signal transduction. We therefore quantitated GS in myocardial membranes obtained before and after chronic exercise in a competitive ELISA based on an antipeptide antibody developed to the alpha S portion of GS. We found a 42% increase in the amounts of GS in right atrial membranes (from 11.4 +/- 0.8 to 16.2 +/- 2.0 pmol/mg; P less than 0.05) and a 76% increase in the amounts of GS in left ventricular membranes (from 15.6 +/- 2.6 to 27.4 +/- 5.2 pmol/mg; P = 0.02) after chronic running. These data suggest that in the heart physiological perturbations can result in changes in the levels of GS, that GS and beta-adrenergic receptor number are not coordinately regulated, and that GS may contribute to altered adrenergic responsiveness independently of changes in beta-adrenergic receptor number.

Animals↗
PubMed1990

Rapid and reversible secretion changes during uncoupling of rat insulin-producing cells.

To determine whether insulin secretion is affected by a blockage of gap junctions between B cells, we have studied the secretion of rat pancreatic islets of Langerhans, primary dispersed islet cells, and cells of the RINm5F line, during short-term exposure to heptanol. Within minutes, this alkanol blocked gap junctions between the B cells of intact islets and abolished their normal secretory response to glucose. These two changes were rapidly and fully reversible after return of the islets to control medium. We further found that heptanol had no significant effect on the glucose-stimulated secretion of single B cells but inhibited that of B cell pairs. In the clone of RINm5F cells, whose junctional coupling and D-glyceraldehyde-induced stimulation of insulin release by aggregated cells were also inhibited by heptanol, this alkanol did not perturb intracellular pH and Ca2+ and the most distal steps of the secretion pathway. In summary, a gap junction blocker affected the secretion of insulin-producing cells by a mechanism which is dependent on cell contact and is not associated with detectable pleiotropic perturbations of the cell secretory machinery. The data provide evidence for the involvement of junctional coupling in the control of insulin secretion.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Effect of central administration of beta-endorphin on lung ornithine decarboxylase activity in developing rats.

Results from a number of studies suggest a role for endogenous opioids in the regulation of lung development and function. Although it is not known which opioid peptides are involved in these processes, accumulated evidence suggests a prominent role for beta-endorphin (BE). Our study examines the effect of BE on lung ornithine decarboxylase (ODC) activity in preweanling rats. ODC catalyzes the rate-limiting step in the synthesis of the polyamines spermidine and spermine, key regulators of cell growth, multiplication, and differentiation. Central (but not peripheral) administration of BE reduced lung ODC activity by as much as 80% in the 6-d-old rat. Significant decreases in ODC activity were seen at doses of BE as low as 0.5 micrograms/g brain wt. In contrast to the reductions in ODC activity, plasma levels of corticosterone in animals administered BE were approximately five times higher than those seen in control animals. BE's actions on ODC activity and plasma corticosterone levels were prevented by naloxone or naltrexone, indicating that both responses are mediated by opioid receptors. Studies of ODC kinetics showed a profound reduction in Vmax (70% below control values), but no change in Km. The effect was observed only during the first 2 wk of postnatal age, a period of time in lung maturation that is characterized by active alveolarization. Because changes in ODC levels during early postnatal life are associated with perturbations in tissue growth and/or function, the data suggest that CNS BE may influence lung maturation through an indirect action that may involve glucocorticoids.

Age Factors↗
PubMed2005

Reaching in children with and without developmental coordination disorder under normal and perturbed vision.

The aim of this study is to describe the reaching action of children with developmental coordination disorder (DCD) and to investigate whether their use of visual feedback during the time course of this action differs from that of normally developing children. Fifty-two children subdivided into 2 age bands (7-8 and 9-10 years) within 2 groups (with and without DCD) participated in this experiment. They were asked to reach for a target positioned either ipsilaterally or contralaterally to the reaching hand in 2 visual conditions: a condition where vision was unrestrained (normal vision) and a condition where they wore glasses with prismatic lenses (perturbed vision). An analysis of the experimental data indicates that the trajectories followed by the DCD group were longer and more curved than those of the control group. Further, the deceleration times were longer for the DCD group than for the normally developing children. The introduction of the prismatic lenses supports the idea that the use of visual feedback by children with DCD may be different from that in children without DCD.

Age Factors↗
PubMed2003

Reactive oxygen disrupts mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis.

Reactive oxygen species (ROS) are involved in a variety of pathophysiological conditions of the testis, and oxidative stress is known to inhibit ovarian and testicular steroidogenesis. The site of ROS-mediated inhibition of steroidogenesis in the corpus luteum and MA-10 tumor Leydig cells was shown to be the hormone-sensitive mitochondrial cholesterol transfer step. The purpose of this study was to examine the effects of ROS on steroidogenic acute regulatory (StAR) protein in MA-10 cells and determine the extent to which MA-10 cell mitochondria are sensitive to oxidative stress. cAMP-stimulated progesterone production was inhibited in a dose-dependent manner in MA-10 cells exposed to H(2)O(2). StAR protein, but not mRNA levels, was decreased in parallel to changes in progesterone production. Even at the highest concentrations of H(2)O(2) tested, there was no effect on P450 side-chain cleavage enzyme protein levels. Oxidative stress from exposure to exogenous xanthine oxidase and xanthine resulted in the inhibition of both progesterone production and StAR protein expression. The mature 30- and 32-kDa intramitochondrial forms of StAR were decreased relative to the 37-kDa extramitochondrial precursor form of StAR, indicating that the ROS-mediated inhibition of StAR protein was due, in part, to the inhibition of mitochondrial import and processing. Vital staining with the fluorescent dye tetramethylrhodamine ethyl ester was used to visualize changes in the mitochondrial electrochemical gradient-dependent membrane potential (Deltapsim). ROS caused a significant dissipation of Deltapsi(m) and time-dependent loss of tetramethylrhodamine ethyl ester fluorescence. The inhibitory effects of H(2)O(2) were transient. There was no evidence for ROS-induced cell death, and following H(2)O(2) removal in the presence of continuous treatment with 8-bromo-cAMP, StAR protein levels and progesterone production were restored. In addition, there was no loss of cell viability following treatment with H(2)O(2) or xanthine/xanthine oxidase as determined by trypan blue exclusion. H(2)O(2) did not cause a significant decrease in total cellular ATP levels. These data indicate that oxidative stress-mediated perturbation of the mitochondria and dissipation of Deltapsi(m) results in the inhibition of StAR protein expression and its import, processing, and cholesterol transfer activity. These findings confirm earlier studies demonstrating the requirement for maintenance of an intact Deltapsi(m) for StAR protein function in cholesterol transport. The significant reduction in the 32- to 30-kDa mature forms of StAR, cessation of cholesterol transport, and loss of Deltapsi(m) are consistent with mitochondrial perturbation because of oxidative stress. This mechanism likely contributes to a host of pathophysiological events evident in testicular disorders such as infection, reperfusion injury, aging, cryptorchidism, and varicocele.

Adenosine Triphosphate↗
PubMed2001

Independent activities of FSH and LH structurally confined in a single polypeptide: selective modification of the relative potencies of the hormones.

The human glycoprotein hormones CG, LH, FSH, and TSH are heterodimers composed of a common alpha subunit noncovalently associated with a hormone-specific beta subunit. Recently, it was reported that a covalently fused triple-domain gonadotropin analog containing FSH beta, CG beta, and alpha subunits was dually active because it bound to both FSH and human CG (hCG)/LH receptors. However, it is not known whether both activities can be uncoupled from each other or whether they change in tandem when modifications are made in the molecule. To address this point, we constructed a triple-domain analog containing FSH beta, LH beta, and alpha subunits, and variants of this analog differing in the carboxyl-terminal region of LH beta. All of the analogs exhibited bifunctional action, i.e. they bound to both LH/hCG and human FSH receptors. FSH binding and signal transduction were similar for all variants and differed less than 2-fold from that of the heterodimer. In contrast, the triple-domain variants manifested distinct individual differences in LH activity. Binding affinity of the longest variant was 30-fold lower than that of the heterodimer. Shortening the length of the LH beta carboxyl-terminal region resulted in decreasing affinities between 210- and more than 480-fold. The potency of adenylate cyclase activation for LH/hCG also decreased as the carboxyl length of LH beta subunit decreased. Thus, while minimally affecting the FSH activity, truncating the carboxyl end of the LH beta subunit in the triple-domain analogs alters the alignment of the LH beta-alpha domains, presumably at the junction between the subunits, and perturbs epitopes required for receptor binding. These data imply that the relative potencies of the two gonadotropin components of a triple-domain structure are independent from each other and can be selectively modified. Because there is a strong rationale for FSH/LH combinations for clinical protocols and patients exhibit variations in metabolic responses in the ratio of FSH/LH, the ability to vary the individual activities represents a potential addition to the therapeutic repertoire for treating infertility.

Animals↗
PubMed2002

In-vitro cytotoxic/cytostatic activity of anionic liposomes containing vinblastine against leukaemic human cell lines.

Liposomes prepared from lipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) with cholesterol were used to investigate the percentage of vinblastine encapsulation and the influence of lipid composition on the retention properties of vinblastine in buffer as well as in cell culture medium. Differential scanning calorimetry (DSC) was applied, to study the effect of cholesterol on the phase transition temperature and on the AH of the two liposome formulations. The cytotoxic and cytostatic activity of the liposome-encapsulated vinblastine was also examined against six leukaemic human cell lines. The results showed that encapsulation of vinblastine into liposomes was greater than 98% with a drug-phospholipid molar ratio of 0.13-0.18. The major improvement in vinblastine retention in buffer as well as in culture medium was achieved by employing DPPG. The DSC data showed that vinblastine exerted a more perturbing effect in DPPC-cholesterol bilayers than in DPPG-cholesterol bilayers and this may explain their lower retention time. The 50% growth-inhibiting (GI50) and cytostatic (TGI) activity of liposomal vinblastine did not seem to be affected by the type of the liposome while the 50% cytotoxic activity (LC50) was affected in four out of the six cell lines tested. The parameters GI50, TGI and LC50 were estimated according to the instructions given by the NCI.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Changes in motor network expression related to moulting behaviour in lobster: role of moult-induced deep hypoxia

The well known rhythmically active pyloric neural network in intact and freely behaving lobsters Homarus gammarus was monitored prior to and following ecdysis. Despite long-lasting hormonal and metabolic alterations associated with this process, spontaneous pyloric network activity remained largely unaltered until the last 12-48 h before exuviation. At this time, the most notable change was a progressive lengthening of pyloric cycle period, which eventually attained 500-600 % of control values. It was only in the very last minutes before ecdysis that burst patterning became irregular and the otherwise strictly alternating motor sequence broke down. After the moult, coordinated rhythmicity was re-established within 10 min. Concomitant with these final changes in motor network expression at ecdysis was a drastic reduction in blood oxygen levels which led to a temporary near-anoxia. By imposing similarly deep hypoxic conditions both on intermoult animals and on the pyloric network in vitro, we mimicked to a large extent the moult-induced changes in pyloric network performance. Our data suggest that, despite major surrounding physiological perturbations, the pyloric network in vivo retains stable pattern-generating properties throughout much of the moulting process. Moreover, some of the most significant modifications in motor expression just prior to ecdysis can be related to a substantial reduction in oxygen levels in the blood.

Journal Article↗
PubMed2005

Virally mediated increased neurotensin 1 receptor in the nucleus accumbens decreases behavioral effects of mesolimbic system activation.

Dopamine receptor agonist and NMDA receptor antagonist activation of the mesolimbic dopamine system increases locomotion and disrupts prepulse inhibition of the acoustic startle response (PPI), paradigms frequently used to study both the pharmacology of antipsychotic drugs and drugs of abuse. In rats, virally mediated overexpression of the neurotensin 1 (NT1) receptor in the nucleus accumbens antagonized d-amphetamine- and dizocilpine-induced PPI disruption, hyperlocomotion, and D-amphetamine-induced rearing. The NT receptor antagonist SR 142948A [2-[[5-(2,6-dimethoxyphenyl)-1-(4-N-(3-dimethylaminopropyl)-N-methylcarbamoyl)-2-isopropylphenyl)-1H-pyrazole-3-carbonyl]amino] adamantane-2-carboxylic acid, hydrochloride] blocked inhibition of dizocilpine-induced hyperlocomotion mediated by overexpression of the NT1 receptor. Together, these results suggest that increased nucleus accumbens NT neurotransmission, via the NT1 receptor, can decrease the effects of activation of the mesolimbic dopamine system and disruption of the glutamatergic input from limbic cortices, resembling the action of the atypical antipsychotic drug clozapine. In contrast to clozapine, virally mediated overexpression of the NT1 receptor in the nucleus accumbens had prolonged protective effects (up to 4 weeks after viral injection) without perturbing baseline PPI and locomotor behaviors. These data further confirm the NT1 receptor as the receptor mediating the antistimulant- and antipsychotic-like properties of NT and provide rationale for the development of NT1 receptor agonists as novel antipsychotic drugs. In addition, the NT1 receptor vector might be a valuable tool for understanding the mechanism of action of antipsychotic drugs and drugs of abuse and may have potential therapeutic applications.

Animals↗
PubMed2005

Structure and energetics of clustered damage sites.

Quantum calculations on duplex DNA trimers were used to model the changes in structure, hydrogen bonding, stacking properties, and electrostatic potential induced by oxidized purine bases and abasic (AP) sites. Results for oxidized purine bases were consistent with experimental data that show small structural and energetic perturbations induced by isolated 8-oxoguanine (8oG). Watson-Crick base pairing was preserved, and no major distortions of the backbone were induced. The thermal destabilization of DNA induced by 8oG was comparable to the energy of a single hydrogen bond. In contrast, AP sites caused substantial distortions of the DNA backbone that were accompanied by relocation of counterions. The loss of Watson-Crick hydrogen bonds in AP sites had the potential to destabilize DNA by 10-20 kcal/mol (0.4-0.8 eV); however, new inter- and intrastrand hydrogen bonds formed after removal of a nucleic acid base that significantly affected the energy of AP sites and introduced a strong dependence on sequence context. Quantum calculations on small DNA fragments provided starting conformations and force-field parameters for classical molecular dynamics simulations of radiation-induced single-strand breaks that most often combine hydrolysis of a phosphate-oxygen (P-O) bond with an AP site and fully or partially degraded sugar ring. P-O bond hydrolysis increased the freedom in backbone torsion angles, which allowed the broken strand to compress and partially fill the hole in the DNA created by the AP site. Results for strand breaks with a 3'phosphoglycolate were similar to those with phosphate end groups.

DNA↗
PubMed2002

Receptor for advanced glycation end products on human synovial fibroblasts: role in the pathogenesis of dialysis-related amyloidosis.

An important component of amyloid fibrils in dialysis-related amyloidosis (DRA) is beta(2)-microglobulin (beta(2)m) modified with advanced glycation end products (AGE). The amyloid deposits are located principally in joint structures, with adjacent chronic inflammatory reaction characterized by monocyte infiltration. This study examined the interaction of AGE-beta(2)m with human synovial fibroblasts and investigated the proinflammatory effects of that interaction. It was demonstrated that human synovial fibroblasts constitutively expressed the receptor for AGE (RAGE). RAGE expression was detected mainly in synovial intima and was upregulated in DRA synovium. (125)I-AGE-beta(2)m bound to immobilized human synovial fibroblasts in a specific, dose-dependent manner (K(d) of approximately 138.0 nM), and binding was inhibited by anti-RAGE IgG. Incubation of human synovial fibroblasts with AGE-beta(2)m induced degradation of this AGE-modified protein, as well as increased monocyte chemoattractant protein-1 (MCP-1) mRNA and protein expression. The amount of MCP-1 produced by AGE-beta(2)m-stimulated human synovial fibroblasts was sufficient to induce the chemotaxis of monocytes. MCP-1 synthesis resulted from engagement of RAGE, because the increase in MCP-1 synthesis was attenuated by preincubation of human synovial fibroblasts with anti-RAGE IgG. These data provide evidence of RAGE-mediated perturbation of human synoviocytes, which may be involved in the pathogenesis of inflammatory processes associated with DRA.

Amyloidosis↗
PubMed2006

Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo.

Hyperglycemia is associated with increased susceptibility to atherothrombotic stimuli. The glycocalyx, a layer of proteoglycans covering the endothelium, is involved in the protective capacity of the vessel wall. We therefore evaluated whether hyperglycemia affects the glycocalyx, thereby increasing vascular vulnerability. The systemic glycocalyx volume was estimated by comparing the distribution volume of a glycocalyx permeable tracer (dextran 40) with that of a glycocalyx impermeable tracer (labeled erythrocytes) in 10 healthy male subjects. Measurements were performed in random order on five occasions: two control measurements, two measurements during normoinsulinemic hyperglycemia with or without N-acetylcysteine (NAC) infusion, and one during mannitol infusion. Glycocalyx measurements were reproducible (1.7 +/- 0.2 vs. 1.7 +/- 0.3 l). Hyperglycemia reduced glycocalyx volume (to 0.8 +/- 0.2 l; P < 0.05), and NAC was able to prevent the reduction (1.4 +/- 0.2 l). Mannitol infusion had no effect on glycocalyx volume (1.6 +/- 0.1 l). Hyperglycemia resulted in endothelial dysfunction, increased plasma hyaluronan levels (from 70 +/- 6 to 112 +/- 16 ng/ml; P < 0.05) and coagulation activation (prothrombin activation fragment 1 + 2: from 0.4 +/- 0.1 to 1.1 +/- 0.2 nmol/l; d-dimer: from 0.27 +/- 0.1 to 0.55 +/- 0.2 g/l; P < 0.05). Taken together, these data indicate a potential role for glycocalyx perturbation in mediating vascular dysfunction during hyperglycemia.

Acetylcysteine↗
PubMed1994

Immunotoxicity of 2,4-diaminotoluene in female B6C3F1 mice.

2,4-Diaminotoluene (DAT) has been demonstrated to be a potent carcinogen. The present studies were carried out to determine the toxic and immunotoxic potential of DAT. Mice exposed to DAT at 25-100 mg/kg per day for 14 days by gavage showed a 42% increase in liver weight and a slight decrease in spleen weight. Histopathologic evaluation of selected organs showed the liver to be the major target with morphological changes which were dose dependent. The high dose (100 mg/kg) was associated with moderate centrilobular necrosis. No abnormal structure was noted in the spleen, lungs, thymus, kidney or mesenteric lymph nodes. The liver toxicity was associated with an elevation in alanine aminotransferase activity. The only change noted in selected hematologic parameters was a 64% increase in peripheral blood leukocytes. Mice exposed to DAT showed a decreased IgM and IgG response to sheep erythrocytes. The decrease was not a function of a decreased number of B cells because the number of B cells increased dose dependently. Proliferative capacity of immunocompetent cells was not impaired by exposure to DAT as measured by the response to several mitogens. The delayed hypersensitivity response to keyhole limpet hemocyanin in mice exposed to DAT was increased. Natural killer cell activity was decreased dose dependently and may represent a spleen cell pool shift because the number of B cells increased in the presence of a decreasing spleen size. Serum C3 was suppressed at the high dose of DAT. Phagocytosis by splenic macrophages, but not peritoneal macrophages, was inhibited by DAT exposure. DAT exposure for 14 days decreased host resistance to the bacteria, Streptococcus pneumoniae and Listeria monocytogenes, while host resistance to the pulmonary tumor model, B16F10, and the PYB6 fibrosarcoma was unaffected by DAT exposure. These data indicate that DAT is hepatotoxic and perturbs the differentiation and maturation of leukocytes.

Administration, Oral↗
PubMed1997

Transcytosis of albumin in endothelial cells is brefeldin A--independent.

To determine whether in endothelial cells (EC) the pathways of endocytosis and transcytosis of macromolecules interconnect, the effect of Brefeldin A (BFA) on these processes was tested. To this purpose EC were grown to confluence on plastic culture dishes or on cell culture chamber inserts placed into corresponding wells, so as to obtain a dual chamber system. The cells maintained the typical characteristics of EC and had an electrical resistance in the range of 30-60 Ohm.cm2. Transendothelial transport of albumin conjugated to the fluorochrom Texas Red (Alb-TR) and of horseradish peroxidase (HRP) added to the upper compartment, in the absence or presence of BFA (0-25 micrograms/ml), was evaluated in aliquots collected from the lower compartment. At different time intervals, quantitative data were obtained by fluorimetry and spectrophotometry. In other experiments transcytosis of Alb-TR was examined in the presence of 100 microM forskolin (an inhibitor of BFA effect). The endocytosis of Alb-TR and HRP was evaluated by incubating EC with the probes, and the internalized tracers determined in the cell lysate using the methods described above. The results showed that BFA has no significant effect on transcytosis of albumin and HRP. In contradistinction, BFA (5 micrograms/ml) reduced markedly endocytosis of HRP (by 47%). Forskolin has no effect on transcytosis. The data indicate that the BFA-induced perturbance in the endocytic route does not affect the transcytotic pathway of albumin, and suggest that in EC, transcytosis of macromolecules may represent a shortcut for rapid and direct transport of some plasma molecules across the cell.

Animals↗
PubMed2002

Comparative short-term prognostic value of hemostatic and inflammatory markers in patients with non-ST elevation acute coronary syndromes.

BACKGROUND: Recent data show that markers of inflammation, endothelial perturbation as well as activation of the coagulation and fibrinolytic systems are altered in unstable angina. The purpose of this study was to compare the 30-day prognostic value of the indexes of inflammation [interleukin-6 (IL-6)], endothelial activation [von Willebrand factor antigen (vWf)], fibrinolysis [plasminogen activator inhibitor-1 (PAI-1)] and coagulation (F1 + 2), in a consecutive series of patients with non-ST elevation acute coronary syndromes. METHODS: Eighty-eight patients consecutively admitted to the coronary care unit because of chest pain occurring within the previous 24 hours were included in the study. Blood was drawn on admission to the coronary care unit and 72 hours thereafter for the assessment of plasma levels of IL-6, vWf, F1 + 2 and PAI-1. Troponin I serum levels were measured 6 to 12 hours after admission. All patients underwent coronary arteriography. RESULTS: Patients were divided into two groups according to their 30-day outcome: 57 patients (group 1) had an uneventful outcome, whereas 31 patients had an adverse clinical event (4 died, 1 had a Q wave myocardial infarction and 26 had refractory angina). The baseline biochemical variables were similar between group 1 and group 2 patients. Seventy-two hours following admission, an increase in the serum levels of IL-6 was observed in 71% of group 2 patients and in 28% of group 1 patients (p = 0.0001). The other measured variables showed significant changes at 72 hours versus entry only in group 1 patients, and no significant difference between the two groups. The areas under the ROC curves were higher for IL-6 (0.72) than for the other variables (0.58 for F1 + 2, 0.52 for vWf and 0.54 for PAI-1). In a multivariate model, including clinical, angiographic, and biochemical variables, only the change in IL-6 over 72 hours was significantly associated with a worse 30-day outcome (odds ratio 8.472, 95% confidence interval 1.030-69.671). CONCLUSIONS: This study shows that a mounting inflammatory process, as indicated by increasing levels of IL-6 over the first 72 hours after admission, is the most powerful predictor of the 30-day prognosis in patients with non-ST elevation acute coronary syndromes.

Aged↗
PubMed1991

Probing the nature of chromosomal DNA-protein contacts by in vivo footprinting.

Of the various approaches employed to unravel the mechanisms of gene regulation, the method of in vivo footprinting seems likely to be increasingly perceived as indispensable. A clear knowledge of the actual pattern of DNA-protein interactions occurring at a given gene within a cell, gained from data obtained with a minimum of external perturbation, can provide a benchmark against which attempts at in vitro reconstruction of the relevant interactions can be judged. This appears particularly important given our current awareness of the degeneracy displayed by certain DNA sequences in terms of their in vitro ability to separately bind to more than one (sometimes several) species of protein factor present in a nuclear extract. The mutual pursuit of both in vivo and in vitro approaches will likely provide the best route to a detailed molecular description of regulatory interactions. Following the introduction of both improved and novel technical approaches, the possibility of probing chromosomal DNA-protein associations at nucleotide resolution is now well within the capacity of most laboratories. In this article the techniques of, probing reagents used for, and some important results obtained by in vivo footprinting are critically discussed.

Animals↗
PubMed1989

Possible involvement of the CD4 molecule in a late activation event on CD4+ T cell proliferation in the human autologous mixed lymphocyte reaction.

CD4 monoclonal antibody (MoAb) was able to inhibit T cell proliferation induced in an autologous mixed lymphocyte reaction (AMLR). The effect of CD4 MoAb on cellular proliferation appears to be directly exerted on CD4+ T lymphocytes, and to be due to inhibition of a post-activation event, since the CD4+ T cell proliferation that occurs after an activation pulse of 24 h with autologous non-T cells could be inhibited when CD4 MoAb was added after, but not during, the pulse period, and the inhibition of autologous MLR-induced CD4+ T cell proliferation by CD4 MoAb was observed even if the Moab was added as late as 72 h after the initiation of culture. The presence of CD4 MoAb did not affect the production of interleukin 2 (IL-2). CD4 MoAb had, however, an inhibitory effect on the expression of IL-2 receptors, such that addition of exogenous IL-2 at the initiation of culture did not restore the AMLR-induced CD4+ T cell proliferation. These results indicate that the hindrance of the recognition of HLA class II products is not the only target of the CD4 MoAb effect in the autologous MLR. Rather, the binding of CD4 MoAb to CD4+ T cells interferes with a late event because it is capable of abolishing the proliferative activity of fully activated CD4+ T cells. The data are compatible with the idea that perturbation of the CD4 molecules can transmit a negative signal to CD4+ T cells.

Antibodies, Monoclonal↗
PubMed1989

Morning insulin requirements. Critique of dawn and meal phenomena.

Morning insulin resistance has frequently been invoked to explain early-morning increases in both basal and breakfast-associated insulin requirements in diabetic patients. This increase in insulin requirements and plasma glucose from 0600 to 0900, when compared with midnight to 0600, has been termed the dawn phenomenon. We believe that the increased need for insulin in the morning has been misinterpreted. Data are reviewed that suggest the major perturbation overnight is a sleep-associated fall in hepatic glucose output, with a return to basal production rates on arousal in the morning. Moreover, the apparent increased insulin requirement for breakfast compared with lunch or supper (meal phenomenon) appears to be related more to lack of residual insulin effect from a preceding meal than to any putative morning insulin resistance. Thus, we found little evidence to support morning insulin resistance as a cause of either the dawn phenomenon (more appropriately designated the sleep phenomenon) or the meal phenomenon. A proper understanding of these phenomena is essential to the management of diabetic patients receiving insulin.

Blood Glucose↗