Novel DNMT3A germline mutations are associated with inherited Tatton-Brown-Rahman syndrome.

Tatton-Brown-Rahman syndrome (TBRS) was recently described in 13 isolated cases with de novo mutations in the DNMT3A gene. This autosomal dominant condition is characterized by tall stature, intellectual disability and a distinctive facial appearance. Here, we report six cases of inherited TBRS caused by novel DNMT3A germline mutations. The affected individuals belong to two sib-ships: four from an Old Order Amish family in America and two from a French Canadian family in Canada. All of them presented with characteristic features of TBRS, including dysmorphic facial features, increased height, intellectual disability, and variable additional features. We performed clinical exome sequencing and identified two mutations in the DNMT3A gene, a c.2312G>A (p.Arg771Gln) missense mutation in the Amish family and a c.2296_2297delAA (p.Lys766Glufs*15) small deletion in the French Canadian family. Parental DNA analysis by Sanger sequencing revealed that the Amish mutation was inherited from the healthy mosaic father. This study reflects the first cases with inherited TBRS and expands the phenotypic spectrum of TBRS.

Radiation-induced delay of nuclear pore formation.

We have shown that radiation affects the nuclear envelope, a membrane structure-closely associated with DNA. The density of nuclear pores visible on freeze-etch surfaces decreased at a rate of 0.042 (pores/mum2)/100 rad with respect to unirradiated cells. This is interpreted as a radiation-induced delay in development of the nuclear envelope.

The effect of temperature on capacitance changes in an oscillating model membrane.

The electrical properties of model membranes are altered during stretching or pressure pulses. We have used a mechanico-electric transduction model to interpret the temperature dependence of capacitance changes produced in oxidized cholesterol membranes during mechanical oscillation. The relative contribution of the torus and bilayor portions of the membrane to the capacitance change is identified. The difference in elasticity between the bilayer and torus decreases rapidly with decreasing temperature and ultimately the torus becomes as solid as the bilayer portion of the model membrane.

Plasma high density lipoproteins in coronary, cerebral and peripheral vascular disease. The influence of various risk factors.

High density lipoprotein (HDL) cholesterol and the HDL/total cholesterol ratio have been measured in 440 patients with coronary, cerebral or peripheral vascular disease and in 440 matched controls. The patients were subdivided into sex- and age-groups and according to physical activity, smoking, hypertension and non-insulin-dependent and insulin-dependent diabetes mellitus. The average HDL cholesterol level was significantly decreased in all the three groups of localization of ischaemic vascular disease (IVD). Plasma HDL concentration in men was lower than in women in every age-group. Lowest values were measured in patients with cerebral vascular diseases. From among the risk factors supposed to be related to IVD, lack of physical exercise resulted in a decrease of HDL cholesterol and HDL/total cholesterol values. In all the three localizations of IVD cigarette smokers had lower HDL levels than non-smokers. The influence of hypertension on serum HDL concentration was not unidirectional. The coexistence of non-insulin-dependent diabetes and IVD resulted in decreased lipid parameters. The sera of insulin-dependent diabetics had higher HDL contents and higher HDL/total cholesterol ratios than those of non-diabetics in all the three localizations of the vascular disease in men and in women suffering from peripheral vascular disease.

Studies on the occurrence of circulating immune complexes in vascular diseases.

The presence of circulating immune complexes was studied in 347 samples of serum from 212 patients with various vascular diseases. Two quantitative methods (complement-consumption assay and C1q-solubility test) were used for the measurement of the concentration of the complexes. Immune complexes were detected in each group of patients tested (coronary arteriosclerosis, myocardial infarction, cerebral artery sclerosis, arteriosclerosis obliterans, phlebothrombosis, pulmonary infarction). A high proportion of positivity was recorded in myocardial infarction (in 43 patients out of the 94 tested) and in arteriosclerosis obliterans (7 out of 11 cases). The possible pathogenic role of the circulating immune complexes is discussed.

Free and complexed anti-lipoprotein antibodies in vascular diseases.

The cell-mediated immune response against low density lipoproteins (LDL) was demonstrated by the migration inhibition test in patients with various vascular diseases. Anti-high density lipoprotein2 (HDL2) cellular immune response was found only in a few patients. LDL and HDL2 binding factors were detected in about 50% of coronary patients. No significant difference in their occurrence was found between the normolipidemic and hyperlipidemic patients nor between patients with hyperlipidemia type II/b and type IV. On the assumption that lipoproteins may act as auto-antigens by forming immune complexes, the presence of anti-LDL and anti-HDL2 activity was investigated in circulating immune complexes obtained by polyethylene glycol (PEG) precipitation from the sera of coronary patients and controls. Using an ELISA technique, PEG-precipitable anti-LDL activity was detected in 23, 11 and 18% of cases with myocardial infarction, angina pectoris and healthy old subjects, respectively. In the immune complexes obtained from the sera of the healthy young donors no anti-LDL activity was found. Anti-HDL2 activity in the immune complexes was demonstrated only in a few cases from among the patients and elderly persons we investigated.

Modulation by enkephalin analogues and neuroleptics of apomorphine-induced stereotypy and turning behaviour in rats.

The aim of the present investigation was to examine which areas of the brain might mediate the anti-apomorphine action of some opioids, which were found previously to be active upon subcutaneous application. As the first step, the substances were injected intracerebroventricularly or into the nucleus accumbens, a mesolimbic region which is rich in dopamine, and the inhibition of stereotypy induced by apomorphine was quantified. In a separate group of animals (rats with unilateral lesion of the nigra) the antagonism of turning behaviour elicited by apomorphine was measured. Substances examined were morphine, a mu-selective opiate; D-Ala2,Nle5-enkephalin sulphonic acid (ES), a delta-selective opioid peptide; D-Met2,Pro5-enkephalinamide (EA), a highly potent but non-selective opioid; and two dopamine receptor blockers, haloperidol and chlorpromazine, for comparison. Examining the antagonism of turning behaviour induced by apomorphine, the order of potency was EA greater than haloperidol greater than morphine greater than ES approximately equal to chlorpromazine if injections of the substances were intracerebroventricular and EA greater than morphine much greater than haloperidol approximately equal to ES much greater than chlorpromazine when administered into the nucleus accumbens. The order of potency for the suppression of stereotypy induced by apomorphine was EA much greater than haloperidol greater than morphine greater than ES greater than chlorpromazine upon intracerebroventricular application and EA much greater than haloperidol greater than morphine ES greater than chlorpromazine if injected into the nucleus accumbens. The data indicate that endogenous opioids might inhibit the activity of dopamine in brain through the nucleus accumbens.

Effect of apomorphine on the antinociceptive activity of morphine.

Apomorphine pretreatment potentiated the analgesic effect of morphine in a dose-dependent manner both in rats and in mice measured by five different tests (writhing, hot plate, inflamed foot, tail-pinch and tail-flick procedures). Furthermore, apomorphine augmented the antinociceptive activity of morphine in tolerant animals as well. In morphine dependent mice the nalorphine precipitated jumping--a withdrawal symptom--was found inhibited by apomorphine treatment. The results are discussed in the light of the numerous but contradictory data available in the literature.

Comparison of tolerance development and dependence capacities of morphine, beta-endorphin, and [D-Met2, Pro5]-enkephalinamide.

The tolerance-development capacities of beta-endorphin, [D-Met2, Pro5]-enkephalinamide, and morphine were compared in rats, and the dependence capacity of morphine was compared with that of the enkephalin analogue in mice. Tolerance to the analgesic effect, as measured by the tail-flick test, developed somewhat more rapidly in the [D-Met2, Pro5]-enkephalinamide-treated group than in the others. A similar relationship was found for the dependence capacity. Considering that the enkephalin analogue displayed the strongest analgesic activity, the well-known correlation between antinociceptive and tolerance development/dependence capacities of opiates seems to be valid for opioid peptides as well.

Species differences in the relative analgesic potencies of some classical opiates and opioid peptides.

The analgesic ED50 values of some classical morphine congeners (morphine, methadone, fentanyl, azidomorphine) in the rat and mouse tail-flick tests were found to be similar. However, several synthetic derivatives of the natural enkephalins were more potent in mice than in rats. (These analogs contain D-amino acid in position 2 and D- or L-sulfonic (or phosphonic) acid residue in position 5). beta-Endorphin, D-Met2, Pro5-enkephalinamide and two partial agonists showed intermediate interspecies relative potencies. According to the data obtained, similar opiate receptors might mediate the analgesic action of classical opiates in rats and in mice. However, the opiate receptors responsible for the antinociceptive effects of the above mentioned enkephalin analogues must be dissimilar in the two species examined. The results are discussed in terms of the role of mu- and delta-receptors in mediation of the analgesic effect induced by different types of opioids.

Biphasic effects of a potent enkephalin analogue (D-Met2,Pro5)-enkephalinamide and morphine on locomotor activity in mice.

The effects of morphine and a potent enkephalin analogue on spontaneous locomotion have been compared in mice. In doses of 3-10 mg/kg SC both compounds induced a brief reduction of motor activity followed by a period of behavioral hyperactivity. Similar receptorial mechanisms are suggested in mediation of their motor effects.

Effects of D-Met2, Pro5-enkephalinamide on pain tolerance and some cognitive functions in man.

The effects of D-Met2, Pro5-enkephalinamide (EA) on pain tolerance and some cognitive functions have been examined in healthy male volunteers. Dihydrocodeine (DC) was used as reference substance. Applying the submaximum effort tourniquet technique EA (10 mg SC) was found to elevate the pain threshold similarly to DC (20 mg SC). Neither DC nor EA impaired the performance in the symbol cancellation test, which quantitates the intensity of attention. In this assay rather a slight improvement was detected. in addition the short-term memory performance (Wechsler test) was also improved by EA and DC. No alteration was seen in the word fluency test, an indicator of long-term (semantic) memory. The data show that EA not only improves pain tolerance but some of its mental effects are similar to those of a classical morphine congener DC.

Effects of methysergide, bromocriptine and naloxone on prolactin, growth hormone and TSH release induced by D-Met2,Pro5-enkephalinamide in man.

D-Met2,Pro5-enkephalinamide (EA) 10 mg, given SC, induced a dramatic rise in serum prolactin (PRL) and growth hormone (GH) levels in healthy male volunteers. The TSH content was also moderately elevated. Naloxone 0.8 mg administered IV abolished these effects. Bromocriptine 2.5 mg given per os also antagonized EA-induced PRL and TSH release but potentiated the GH surge. Methysergide 2.0 mg administered orally partially reversed EA-elicited PRL release, further augmented GH liberation and did not modify TSH output. The data indicate that inhibition of the dopaminergic tone and/or activation of certain serotonergic mechanisms play an important role in the EA-induced release of PRL and TSH. However, primarily other neurotransmitters might mediate the GH liberation elicited by this opioid peptide.

A comparison of cell killing by heat and/or X rays in Chinese hamster V79 cells, Friend erythroleukemia mouse cells, and human thymocyte MOLT-4 cells.

The radiation and/or heat sensitivity of Chinese hamster V79 cells, Friend erythroleukemia (FELC) mouse cells, and MOLT-4 human transformed thymocytes were compared. MOLT-4 cells were more radiosensitive (D0 = 0.50 Gy) than FELC (D0 = 0.65 Gy) and V79 cells (D0 = 1.43 Gy). Arrhenius analysis showed that MOLT-4 cells were more heat sensitive than FELC or V79 cells below 42.0 degrees C, but more heat resistant at higher temperatures. In addition, the MOLT-4 cells showed a single-heat inactivation energy between 41.0 and 45.0 degrees C, while FELC and V79 cells both showed a transition in the inactivation energy at about 43.0 and 43.5 degrees C, respectively. These differences may be related to the fact that the upper temperature limit for the development of thermal tolerance during continuous heating was lower for MOLT-4 cells than for FELC or V79 cells. Killing of FELC and V79 cells was dependent on the sequence in which heat and X rays were applied, but the greatest effect was obtained when both treatments were given simultaneously. Recovery occurred when treatments were separated by incubation at 37.0 degrees C. The MOLT-4 cells did not show a sequence dependence for heating and irradiation. Survival of MOLT-4 cells after heating and/or irradiation was compared using trypan blue dye exclusion or colony formation. Both assays showed similar qualitative responses, but survival levels measured by the trypan blue assay were much higher than those determined from the colony-forming assay.

The effect of prostaglandin F2 alpha treatment on the endometrial oestradiol receptor system.

The effects of prostaglandin F2 alpha treatment on oestradiol receptor levels were studied in human decidual tissues from the first trimester of pregnancy. Two types of nuclear oestradiol binding sites in decidual tissue were detected. Type I receptors bind the oestradiol with high affinity and low capacity (Kd = 1.4 nM, Bmax = 0.3 pmol/mgDNA, Hillcoeff = 1), while type II binding sites have lower affinity and higher capacity for the hormone (Kd = 20.4 nM, Bmax = 2.0 pmol/mgDNA, Hillcoeff = 3). The nuclear oestrogen binding capacities of decidual tissue were affected by prostaglandin F2 alpha in a time dependent manner. Within the first 10 hrs after prostaglandin F2 alpha application the concentration of low affinity, high capacity binding sites (type II) was enhanced in comparison with the control values. Later, 20-24 hrs after the treatment, both types of nuclear oestrogen binding sites were increased significantly. When prostaglandin F2 alpha treatment failed to induce abortion no change in the concentration of nuclear oestrogen binding sites was observed. Oestradiol receptor concentration in the cytoplasm apparently was not affected by prostaglandin F2 alpha treatment.

Thermoregulatory effects of D-met2-pro5-enkephalinamide.

Changes elicited in the body temperature by subcutaneous injection of the synthetic D-met2-pro5-enkephalinamide (D-met2-pro5-EA) were studied in CFY rats. D-met2-pro5-EA in doses of 8, 20 and 40 mg/kg caused hyperthermia. If the animals were restrained, this hyperthermic effect was attenuated. Ten mg/kg naloxone completely prevented the elevation of body temperature. Previous administration of 300 mg/kg capsaicin in fractionated daily doses resulted in a facilitation of the hyperthermic reaction. In contrast to other opioids, hypothermia was never observed. Our observations support the concept of multiple opiate-sensitive mechanisms in thermoregulation.

Effect of [D-Met2,Pro5]enkephalinamide on gastric ulceration and transmucosal potential difference.

The effects of [D-Met2,Pro5]enkephalinamide, morphine and naloxone have been examined in two different models of experimentally elicited gastric mucosal lesions. One of them was the classic cold-restraint stress-induced ulceration. The other was a less frequently applied procedure, involving the measurement of decreases in the transmucosal potential difference, which is also a sensitive indicator of mucosal damage. The opioid agonists studied, [D-Met2,Pro5]enkephalinamide and morphine, aggravated, whereas naloxone, the pure opiate antagonist, mitigated the lesions in both models. The protective action of naloxone points to an eventual role of endogenous opioids in the generation of these types of mucosal lesions. Morphine is selective ligand of mu-opiate receptors. The enkephalin analogue, however, binds to both mu- and delta-receptors. Therefore, the potent pro-ulcerogenic action of the enkephalin analogue indicates that both the mu- and delta-receptors were involved in these models of experimental gastric lesions. The clarification of the eventual role of kappa-receptors requires further experimental work with a selective ligand of this subtype of opiate receptors.

Apparent antinociceptive and anti-inflammatory effects of GYKI 52466.

GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) was examined in a battery of analgesia and anti-inflammatory tests in rats and mice, respectively. Its 3-N-acetyl (GYKI 53773) and 3-N-methylcarbamoyl (GYKI 53784) derivatives were also examined in some assays. These 2,3-benzodiazepines, known as prototypic non-competitive antagonists of AMPA receptors, showed a peculiar profile in some routinely used antinociceptive tests. They were found fairly potent in rat tail flick and mouse phenylquinone writhing assays but the dose-response curves were rather shallow as compared to that of morphine. Their action is stereoselective, i.e., the (+) isomers were found inactive, in agreement with the previous in vitro studies. Their antinociceptive effect could not be reversed by naloxone and the GYKI compounds did not potentiate significantly the morphine-induced analgesia. In the mouse hot plate assay the 2,3-benzodiazepines were active only in doses inducing visible motor incapacitation. In rats, GYKI 52466 weakly reduced the hypersensitivity accompanying acute carrageenan edema. However, it potently inhibited the hyperalgesia during Freund adjuvant-induced chronic arthritis. In the latter assay GYKI 52466 also attenuated the body weight loss without altering the paw edema. The present findings confirm reports in the literature which indicate AMPA receptors may contribute to certain forms of pathological hyperalgesia, e.g., to that detectable in inflamed tissues.

Naloxone-insensitive modulation of gastric acid output by [D-Met2,Pro5]enkephalinamide in rats.

Studies from our laboratory have previously shown that two opioid agonists (morphine and [D-Met2,Pro5]enkephalinamide) aggravate, whereas naloxone inhibits cold-restraint stress-induced ulceration in rats. In the present study the effects of these substances were examined using the Shay-model. Contrary to expectations, both naloxone and the opioid agonists decreased gastric acid output. Naloxone in combination with either opioid agonist failed to reverse their inhibitory action. Thus the secretory activity of the stomach may be modulated by opioids in both naloxone-reversible and irreversible ways.

Enkephalin-like character and analgesia.

The opioid activities of enkephalin analogues bearing D- or L-aminopentane-sulfonic/phosphonic acid at position 5 were studied in vitro, in electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations and in vivo in the rat tail-flick test. Using their in vitro effects Met-enkephalin-like, beta-endorphin-like, (nor)morphine-like and derivatives of intermediate character could be differentiated. Correlating the in vitro activities with the analgesic activity in vivo it is concluded that the enkephalin-like character in a pentapetide may hinder the expression of analgesic activity, when the compounds are given into the cerebroventricular system.