A recurrent de novo missense mutation in UBTF causes developmental neuroregression.

UBTF (upstream binding transcription factor) exists as two isoforms; UBTF1 regulates rRNA transcription by RNA polymerase 1, whereas UBTF2 regulates mRNA transcription by RNA polymerase 2. Herein, we describe 4 patients with very similar patterns of neuroregression due to recurrent de novo mutations in UBTF (GRCh37/hg19, NC_000017.10: g.42290219C > T, NM_014233.3: c.628G > A) resulting in the same amino acid change in both UBTF1 and UBTF2 (p.Glu210Lys [p.E210K]). Disease onset in our cohort was at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Notable early features included hypotonia with a floppy gait, high-pitched dysarthria and hyperactivity. Later features included aphasia, dystonia, and spasticity. Speech and ambulatory ability were lost by the early teens. Magnetic resonance imaging showed progressive generalized cerebral atrophy (supratentorial > infratentorial) with involvement of both gray and white matter. Patient fibroblasts showed normal levels of UBTF transcripts, increased expression of pre-rRNA and 18S rRNA, nucleolar abnormalities, markedly increased numbers of DNA breaks, defective cell-cycle progression, and apoptosis. Expression of mutant human UBTF1 in Drosophila neurons was lethal. Although no loss-of-function variants are reported in the Exome Aggregation Consortium (ExAC) database and Ubtf-/- is early embryonic lethal in mice, Ubtf+/- mice displayed only mild motor and behavioral dysfunction in adulthood. Our data underscore the importance of including UBTF E210K in the differential diagnosis of neuroregression and suggest that mainly gain-of-function mechanisms contribute to the pathogenesis of the UBTF E210K neuroregression syndrome.

Clinical associations of proinflammatory cytokines, oxidative biomarkers and vitamin D levels in systemic lupus erythematosus.

Background The abnormal biological activity of cytokines plays an important role in the pathophysiology of both systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). Several studies have highlighted the association of vitamin D and certain pro-inflammatory cytokines with disease activity in SLE. However, there are limited data on the association of vitamin D and antiphospholipid antibodies (aPL) with various proinflammatory biomarkers in these patients and their relative impact on clinical outcomes. Methods The serum levels of several aPL, 25-hydroxy-vitamin D, pro-inflammatory cytokines including IFNα, IL-1ß, IL-6, IL-8, IP10, sCD40L, TNFα and VEGF were measured in 312 SLE patients from the Jamaican ( n = 45) and Hopkins ( n = 267) lupus cohorts using commercial Milliplex and ELISA assays. Oxidized LDL/ß2glycoprotein antigenic complexes (oxLß2Ag) and their associated antibodies were also measured in the Jamaican cohort. Healthy controls for oxidative marker and cytokine testing were used. Results Abnormally low vitamin D levels were present in 61.4% and 73.3% of Hopkins and Jamaican SLE patients, respectively. Median concentrations of IP10, TNFα, sCD40L and VEGF were elevated in both cohorts, oxLß2Ag and IL-6 were elevated in the Jamaican cohort, and IFNα, IL-1ß and IL-8 were the same or lower in both cohorts compared to controls. IP10 and VEGF were independent predictors of disease activity, aPL, IP10 and IL-6 were independent predictors of thrombosis and IL-8, and low vitamin D were independent predictors of pregnancy morbidity despite there being no association of vitamin D with pro-inflammatory cytokines. Conclusions Our results indicate that aPL-mediated pro-inflammatory cytokine production is likely a major mechanism of thrombus development in SLE patients. We provide presumptive evidence of the role IL-8 and hypovitaminosis D play in obstetric pathology in SLE but further studies are required to characterize the subtle complexities of vitamin D's relationship with cytokine production and disease activity in these patients.

Interactive effects between water temperature, microparticle compositions, and fiber types on the marine keystone species Americamysis bahia.

Recently, there has been an increasing emphasis on examining the ecotoxicological effects of anthropogenic microparticles (MPs), especially microplastic particles, and related issues. Nevertheless, a notable deficiency exists in our understanding of the consequences on marine organisms, specifically in relation to microfibers and the combined influence of MPs and temperature. In this investigation, mysid shrimp (Americamysis bahia), an important species and prey item in estuarine and marine food webs, were subjected to four separate experimental trials involving fibers (cotton, nylon, polyester, hemp; 3 particles/ml; approximately 200 µm in length) or fragments (low-density Polyethylene: LDPE, polylactic acid: PLA, and their leachates; 5, 50, 200, 500 particles/ml; 1-20 µm). To consider the effects in the context of climate change, three different temperatures (22, 25, and 28 °C) were examined. Organismal growth and swimming behavior were measured following exposure to fragments and microfibers, and reactive oxygen species and particle uptake were investigated after microfiber exposure. To simulate the physical characteristics of MP exposure, such as microfibers obstructing the gills, we also assessed the post-fiber-exposure swimming behavior in an oxygen-depleted environment. Data revealed negligible fragment, but fiber exposure effects on growth. PLA leachate triggered higher activity at 25 °C and 28 °C; LDPE exposures led to decreased activity at 28 °C. Cotton exposures led to fewer behavioral differences compared to controls than other fiber types. The exposure to hemp fibers resulted in significant ROS increases at 28 °C. Microfibers were predominantly located within the gastric and upper gastrointestinal tract, suggesting extended periods of residence and the potential for obstructive phenomena over the longer term. The combination of increasing water temperatures, microplastic influx, and oxidative stress has the potential to pose risks to all components of marine and aquatic food webs.

The nitric oxide redox sibling nitroxyl partially circumvents impairment of platelet nitric oxide responsiveness.

Impaired platelet responsiveness to nitric oxide (NO resistance) is a common characteristic of many cardiovascular disease states and represents an independent risk factor for cardiac events and mortality. NO resistance reflects both scavenging of NO by superoxide (O2(-)), and impairment of the NO receptor, soluble guanylate cyclase (sGC). There is thus an urgent need for circumvention of NO resistance in order to improve clinical outcomes. Nitroxyl (HNO), like NO, produces vasodilator and anti-aggregatory effects, largely via sGC activation, but is not inactivated by O2(-). We tested the hypothesis that HNO circumvents NO resistance in human platelets. In 57 subjects with or without ischemic heart disease, platelet responses to the HNO donor isopropylamine NONOate (IPA/NO) and the NO donor sodium nitroprusside (SNP) were compared. While SNP (10µM) induced 29±3% (p<0.001) inhibition of platelet aggregation, IPA/NO (10µM) caused 75±4% inhibition (p<0.001). In NO-resistant subjects (n=28), the IPA/NO:SNP response ratio was markedly increased (p<0.01), consistent with partial circumvention of NO resistance. Similarly, cGMP accumulation in platelets was greater (p<0.001) with IPA/NO than with SNP stimulation. The NO scavenger carboxy-PTIO (CPTIO, 200µM) inhibited SNP and IPA/NO responses by 92±7% and 17±4% respectively (p<0.001 for differential inhibition), suggesting that effects of IPA/NO are only partially NO-mediated. ODQ (10µM) inhibited IPA/NO responses by 36±8% (p<0.001), consistent with a contribution of sGC/haem to IPA/NO inhibition of aggregation. There was no significant relationship between whole blood ROS content and IPA/NO responses. Thus the HNO donor IPA/NO substantially circumvents platelet NO resistance while acting, at least partially, as a haem-mediated sGC activator.

Micron-size tire tread particles leach organic compounds at higher rates than centimeter-size particles: Compound identification and profile comparison.

Tire tread particles (TTP) are environmentally prevalent microplastics and generate toxic aqueous leachate. We determined the total carbon and nitrogen leachate concentrations and chemical profiles from micron (∼32 µm) and centimeter (∼1 cm) TTP leachate over 12 days. Dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were used to measure the concentration of leached compounds. Nontargeted chemical analysis by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/TOF-MS) was used to compare the chemical profiles of leachates. After leaching for 12 days, DOC was 4.0 times higher in the micron TTP leachate than in the centimeter TTP leachate, and TDN was 2.6 times higher. The total GC×GC/TOF-MS chromatographic feature peak area was 2.9 times greater in the micron TTP leachate than the centimeter TTP leachate, and similarly, the total relative abundance of 54 tentatively identified compounds was 3.3 times greater. We identified frequently measured tire-related chemicals, such as 6PPD, N-cyclohexyl-N'-phenylurea (CPU), and hexa(methoxymethyl)melamine (HMMM), but nearly 50% of detected chemicals were not previously reported in tire literature or lacked toxicity information. Overall, the results demonstrate that smaller TTP have a greater potential to leach chemicals into aquatic systems, but a significant portion of these chemicals are not well-studied and require further risk assessment.

Interpreting the biological relevance of bioinformatic analyses with T-DNA sequence for protein allergenicity.

Global regulatory agencies require bioinformatic sequence analysis as part of their safety evaluation for transgenic crops. Analysis typically focuses on encoded proteins and adjacent endogenous flanking sequences. Recently, regulatory expectations have expanded to include all reading frames of the inserted DNA. The intent is to provide biologically relevant results that can be used in the overall assessment of safety. This paper evaluates the relevance of assessing the allergenic potential of all DNA reading frames found in common food genes using methods considered for the analysis of T-DNA sequences used in transgenic crops. FASTA and BLASTX algorithms were used to compare genes from maize, rice, soybean, cucumber, melon, watermelon, and tomato using international regulatory guidance. Results show that BLASTX for maize yielded 7254 alignments that exceeded allergen similarity thresholds and 210,772 alignments that matched eight or more consecutive amino acids with an allergen; other crops produced similar results. This analysis suggests that each nontransgenic crop has a much greater potential for allergenic risk than what has been observed clinically. We demonstrate that a meaningful safety assessment is unlikely to be provided by using methods with inherently high frequencies of false positive alignments when broadly applied to all reading frames of DNA sequence.

Internalization, reduced growth, and behavioral effects following exposure to micro and nano tire particles in two estuarine indicator species.

Synthetic rubber emissions from automobile tires are common in aquatic ecosystems. To assess potential impacts on exposed organisms, early life stages of the estuarine indicator species Inland Silverside (Menidia beryllina) and mysid shrimp (Americamysis bahia) were exposed to three tire particle (TP) concentrations at micro and nano size fractions (0.0038, 0.0378 and 3.778 mg/L in mass concentrations for micro size particles), and separately to leachate, across a 5-25 PSU salinity gradient. Following exposure, M. beryllina and A. bahia had significantly altered swimming behaviors, such as increased freezing, changes in positioning, and total distance moved, which could lead to an increased risk of predation and foraging challenges in the wild. Growth for both A. bahia and M. beryllina was reduced in a concentration-dependent manner when exposed to micro-TP, whereas M. beryllina also demonstrated reduced growth when exposed to nano-TP (except lowest concentration). TP internalization was dependent on the exposure salinity in both taxa. The presence of adverse effects in M. beryllina and A. bahia indicate that even at current environmental levels of tire-related pollution, which are expected to continue to increase, aquatic ecosystems may be experiencing negative impacts.

Bacillus anthracis spore suspensions: determination of stability and comparison of enumeration techniques.

AIM: To determine the stability and variability in concentration of spore suspensions of Bacillus anthracis (BA) spore suspensions by comparing different methods of enumeration and to detect changes, if any, under different storage conditions. METHODS AND RESULTS: Plate and microscope counts were compared to measuring the genomic equivalents based on DNA content BA spore suspensions. We developed chemical methods to extract spore DNA and extra-spore (ES) DNA. DNA mass was determined by gel electrophoresis and QPCR assays were developed using the markers on the chromosome (rpoB) and the pXO1 plasmid (pag). The plate counts and microscope counts were very stable (for up to 900 days). The effect of freezing and the presence of additives in samples were tested for up to 300 days, and the results indicated that the additives tested and freezing did not decrease the viability or microscope counts. CONCLUSIONS: Bacillus anthracis spore suspensions can be stored for long periods of time without significant loss of viability or clumping. The content of ES DNA was variable and changed with time. SIGNIFICANT AND IMPACT OF THE STUDY: The study shows that BA spore suspensions can be developed for reference materials providing a uniform basis for comparing detection equipment and results from different laboratories.

Complement-independent adherence of Escherichia coli to complement receptors in vitro.

We studied adherence to human cells by a strain of Escherichia coli. Adherence to erythrocytes was assessed directly by phase-contrast microscopy and indirectly by hemagglutination; adherence to peripheral blood leukocytes, using radiolabeled bacteria and subsequent determination of leukocyte-associated radioactivity; and adherence to renal glomeruli, by microscopy of fluoresceinated bacteria and of Gram-stained nonfluoresceinated bacteria. In serum-free systems, E. coli of this strain adhered to human erythrocytes, which have surface receptors for the third component of complement (C3), but not to erythrocytes from species lacking this receptor. 1 mM trypan blue, a reagent that inhibits complement receptor function, inhibited adherence to human erythrocytes, as well as adherence to leukocytes and glomeruli. Preincubation of erythrocytes and leukocytes with complement-coated zymosan particles partially blocked subsequent bacterial adherence. Incubation of human erythrocytes with aging human serum, with trypsin-cleaved C3, or with C3 cleaved by the classical pathway convertase (EAC142)-all of which treatments deposited C3 on the erythrocyte surface, presumably at C3 receptors-inhibited subsequent E. coli adherence. Finally, incubation of E. coli with rabbit antiserum to human C3 blocked adherence to erythrocytes.Bacterial hemagglutination and erythrocyte adherence were not inhibited by mannose in concentrations up to 2.5%. And this strain of E. coli did not adhere to or agglutinate guinea pig erythrocytes, the usual test particle used for demonstration of common pili. Finally, electron microscopy of adherent bacteria showed only rare surface pili. In contrast, adherence to and agglutination of guinea pig erythrocytes by a stock piliated E. coli was inhibited by mannose but not by trypan blue. We conclude that organisms of this strain of E. coli adhere to human erythrocytes, leukocytes, and glomeruli at complement receptors. Complement is not required for this interaction. Adherence apparently involves a C3-like structure on the bacterial surface, but bacterial surface pili play no role. The physiological or pathological role of this adherence is not apparent, but study of this phenomenon may elucidate functions of complement receptors on various cells.

Green fluorescent protein as a marker for expression of a second gene in transgenic plants.

The use of transgenic crops has generated concerns about transgene movement to unintended hosts and the associated ecological consequences. Moreover, the in-field monitoring of transgene expression is of practical concern (e.g., the underexpression of an herbicide tolerance gene in crop plants that are due to be sprayed with herbicide). A solution to these potential problems is to monitor the presence and expression of an agronomically important gene by linking it to a marker gene, such as GFP. Here we show that GFP fluorescence can indicate expression of the Bacillus thuringiensus cry1Ac gene when co-introduced into tobacco and oilseed rape, as demonstrated by insect bioassays and western blot analysis. Furthermore we conducted two seasons of field experiments to characterize the performance of three different GFP genes in transgenic tobacco. The best gene tested was mGFP5er, a mutagenized GFP gene that is targeted to the endoplasmic reticulum. We also demonstrated that host plants synthesizing GFP in the field suffered no fitness costs.

Induction of covalent DNA modifications and micronucleated erythrocytes by 4-nitroquinoline 1-oxide in adult and fetal mice.

Pregnancy and development are known to modify carcinogenesis. Little is known about the mechanism for the modulation. These studies investigated the relative sensitivity of nonpregnant, pregnant, and fetal mice to the induction of covalent DNA modifications and micronucleated erythrocytes by 4-nitroquinoline 1-oxide (4-NQO). Our results revealed that 4-NQO was bound to guanine nucleotides of DNA in all maternal and fetal organs tested. The adduct levels ranged from 2-60 base modifications per 10(9) DNA bases when 4-NQO was administered s.c. Overall, 4-NQO bound preferentially to DNA of the maternal tissues compared with that of the corresponding fetal tissues, with the exception of the liver. The adduct levels in maternal and fetal organs fell into 3 distinct levels. The greatest binding was in maternal lungs and pancreas (the target organs for carcinogenesis). The lowest binding levels were in maternal liver and all fetal organs studied. Gestation age at the time of 4-NQO treatment did not produce a significant effect on the amounts of adduct formation in the tissues examined, with the exception of placenta and bone marrow. Chronic treatment did not affect binding preference. At the cellular level, 4-NQO treatment induced twice the frequency of micronucleated erythrocytes in the bone marrow of pregnant mice compared with the nonpregnant mice and fetal liver, on a mg/kg basis. However, the polychromatic erythrocytes of fetal liver were more sensitive than those of adult bone marrow to the induction of micronuclei, when adduct levels were taken into account. A positive correlation of organotropsim between 4-NQO-induced DNA adducts and carcinogenicity was observed for maternal tissues, but not for fetal tissues. Fetal tissues, overall, lack the enzymes to metabolically activate 4-NQO. Fetal cells elicit greater biological responses, compared with adult cells, at equal adduct levels. This study reveals that the effective doses in maternal and fetal tissues may differ and, therefore, will be a better basis for further understanding the molecular mechanism of transplacental carcinogenesis.

Low dose dietary nitrate improves endothelial dysfunction and plaque stability in the ApoE-/- mouse fed a high fat diet.

BACKGROUND: Nitric oxide (NO) is an important vascular signalling molecule. NO is synthesised endogenously by endothelial nitric oxide synthase (eNOS). An alternate pathway is exogenous dietary nitrate, which can be converted to nitrite and then stored or further converted to NO and used immediately. Atherosclerosis is associated with endothelial dysfunction and subsequent lesion formation. This is thought to arise due to a reduction in the bioavailability and/or bioactivity of endogenous NO. AIM: To determine if dietary nitrate can protect against endothelial dysfunction and lesion formation in the ApoE-/- mouse fed a high fat diet (HFD). METHODS AND RESULTS: ApoE-/- fed a HFD were randomized to receive (i) high nitrate (10mmol/kg/day, n=12), (ii) moderate nitrate (1mmol/kg/day, n=8), (iii) low nitrate (0.1mmol/kg/day, n=8), or (iv) sodium chloride supplemented drinking water (control, n=10) for 10 weeks. A group of C57BL6 mice (n=6) received regular water and served as a healthy reference group. At 10 weeks, ACh-induced vessel relaxation was significantly impaired in ApoE-/- mice versus C57BL6. Mice supplemented with low or moderate nitrate showed significant improvements in ACh-induced vessel relaxation compared to ApoE-/- mice given the high nitrate or sodium chloride. Plaque collagen expression was increased and lipid deposition reduced following supplementation with low or moderate nitrate compared to sodium chloride, reflecting increased plaque stability with nitrate supplementation. Plasma nitrate and nitrite levels were significantly increased in all three groups fed the nitrate-supplemented water. CONCLUSION: Low and moderate dose nitrate significantly improved endothelial function and atherosclerotic plaque composition in ApoE-/- mice fed a HFD.

Inflammasome activity is essential for one kidney/deoxycorticosterone acetate/salt-induced hypertension in mice.

BACKGROUND AND PURPOSE: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1ß and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1ß and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH: Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. KEY RESULTS: 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1ß, as well as protein levels of active caspase-1 and mature IL-1ß. Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. CONCLUSIONS AND IMPLICATIONS: Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1ß pathway as a potential therapeutic target in hypertension.

Mechanical and microstructural properties of "wet" alginate and composite films containing various carbohydrates.

Composite "wet" alginate films were manufactured from alginate-carbohydrate solutions containing 5% alginate and 0.25% pectin, carrageenan (kappa or iota), potato starch (modified or unmodified), gellan gum, or cellulose (extracted or commercial). The "wet" alginate films were used as a model to understand co-extruded alginate sausage casings that are currently being used by several sausage manufacturers. The mechanical, optical, and microstructural properties of the calcium cross-linked composite films were explored. In addition, the water holding capacity and textural profile analysis properties of the alginate-carbohydrate gels were studied. The results indicate that the mechanical properties of "wet" alginate films/casings can be modified by adding various carbohydrates to them. Alginate films with pectin, carrageenan, and modified potato starch had significantly (P < 0.05) greater elongation values than pure alginate films. The alginate-pectin films also had greater (P < 0.05) tensile strengths than the pure alginate films. Alginate films with extracted cellulose, commercial cellulose, and modified potato starch had lower (P < 0.05) puncture force, distance, and work values than the alginate control films. Transmission electron microscopy images showed a very uniform alginate network in the control films. Several large cellulose fibers were visible in the films with extracted cellulose, while the cellulose fibers in the films with commercial cellulose were difficult to distinguish. Despite these apparent differences in cellulose fiber length, the 2 cellulose films had similar puncture and tensile properties.

NOX1 deficiency in apolipoprotein E-knockout mice is associated with elevated plasma lipids and enhanced atherosclerosis.

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are enzymes that generate reactive oxygen species (ROS). NOX2 activity in the vascular wall is elevated in hypercholesterolemia, and contributes to oxidative stress and atherogenesis. Here we examined the role of another NOX isoform, NOX1, in atherogenesis in apolipoprotein E-knockout (APOE(-/-)) mice fed a Western diet for 14 weeks. Although NOX1 mRNA expression was unchanged in aortas from APOE(-/-) versus wild-type mice, expression of the NOX1-specific organizer, NOXO1, was diminished, consistent with an overall reduction in NOX1 activity in APOE(-/-) mice. To examine the impact of a further reduction in NOX1 activity, APOE(-/-) mice were crossed with NOX1(-/y) mice to generate NOX1(-/y)/APOE(-/-) double-knockouts. NOX1 deficiency in APOE(-/-) mice was associated with 30-50% higher plasma very-low-density lipoprotein (VLDL)/LDL and triglyceride levels (P < 0.01). Vascular ROS levels were also elevated by twofold in NOX1(-/y)/APOE(-/-) versus APOE(-/-) mice (P < 0.05), despite no changes in expression of other NOX subunits. Although en face analysis of the descending aorta revealed no differences in plaque area between NOX1(-/y)/APOE(-/-) and APOE(-/-) mice, intimal thickening in the aortic sinus was increased by 40% (P < 0.05) in the double-knockouts. Moreover, NOX1 deficiency was associated with a less stable plaque phenotype; aortic sinus lesions contained 60% less collagen (P < 0.01), 40% less smooth muscle (P < 0.01), and 2.5-fold higher levels of matrix metalloproteinase-9 (P < 0.001) than lesions in APOE(-/-) mice. Thus, these data, which suggest a protective role for NOX1 against hyperlipidemia and atherosclerosis in APOE(-/-) mice, highlight the complex and contrasting roles of different NOX isoforms (e.g., NOX2 versus NOX1) in vascular pathology.

Pathologic correlates of increased signals of the centrum ovale on magnetic resonance imaging.

The pathologic correlates of increased signal in the white matter of the centrum ovale in postmortem magnetic resonance imaging were investigated in an unselected series of 15 autopsies. Two types of magnetic resonance imaging hyperintensities could be separated on the basis of size (10-mm cutoff): extensive and punctate. The pathologic basis of extensive hyperintensities was large areas of pallor with ill-defined margins, located in the central white matter and sparing the subcortical U fibers on both myelin and axonal stains. Microscopically, these areas showed diffuse vacuolation and significant reduction in the areal densities of glial cells. This change was never seen in areas that did not show extensive white matter hyperdensities on magnetic resonance imaging. The correlates of punctate magnetic resonance imaging hyperintensities were less well defined; dilated Virchow-Robin spaces probably represent a common cause of this phenomenon.

Presynaptic alpha-adrenoceptor blocking properties among tri- and tetra-cyclic antidepressant drugs.

1 The effect of various antidepressants (5 x 10(-8) to 2 x 10(-5) M) on the resting overflow of tritium, on the evoked overflow and the contractile response to electrical stimulation (2.5 Hz, 2.0 ms) has been determined in mouse vas deferens previously incubated with [(3)H]-(-)-noradrenaline.2 Mianserin and ORG GC 94 produced a concentration-dependent increase of more than two fold in the electrically evoked overflow and the contractile response and, at the highest concentration, slightly increased resting release. These effects were largely unchanged in the presence of a concentration of cocaine effective in blocking noradrenaline uptake (1.1 x 10(-5) M).3 The ability of phentolamine (1 x 10(-5) M) to increase both the evoked overflow of tritium and the contractile response was greatly reduced when these parameters were already elevated by the presence of mianserin or ORG GC 94.4 The inhibitory effect of exogenous (-)-noradrenaline on evoked overflow was greatly reduced in the presence of mianserin or ORG GC 94 (4 x 10(-6) M).5 The inhibitory effect of clonidine on the twitch response of the mouse vas deferens was antagonized by mianserin and ORG GC 94 in a competitive manner (pA(2) values 7.3 and 7.1 respectively).6 Maprotiline, desipramine and nortriptyline (> 3 x 10(-6) M) produced a parallel fall in both evoked tritium overflow and in the contractile response and increased the resting overflow at higher concentrations. These effects were largely unchanged in the presence of cocaine (1.1 x 10(-5) M).7 Doxepin, imipramine and iprindole all increased resting overflow at high concentrations (2 x 10(-5) M) but produced only small changes in evoked overflow and in the contractile response at lower concentrations.8 It is concluded that mianserin and ORG GC 94 produce a blockade of presynaptic alpha-adrenoceptors which could contribute to an antidepressant effect but that this type of action is not common to all antidepressants.

The effect of stimulus intensity on alpha-adrenoceptor-mediated feedback control of noradrenaline release.

1 Mouse vas deferns stimulated transmurally (2.5 Hz, 2-32 V, 40-620 mA, FOR 45 s) responded with a twitch and a secondary contraction. Both responses were abolished by cinchocaine and were voltage-dependent. 2 In tissues previously incubated with (3H)-(--)-noradrenaline, stimulation also produced an increase in tritium overflow from the tissue. Phentolamine increased tritium overflow by 19% at high stimulus intensities (30 V, 600 mA) and by 130% at low stimulus intensities (11 V, 200 mA). 3 It is concluded that alpha-adrenoceptor-mediated feedback control of noradrenaline release is more marked at low stimulus intensities and that this is compatible with a role for calcium ions in this control mechanism.

A comparison in rabbit isolated hearts of the dysrhythmogenic potential of amitriptyline, maprotiline and mianserin in relation to their ability to block noradrenaline uptake.

1. In isolated hearts of rabbits, perfusion with (-)-noradrenaline (0.0059 to 5.9 micronM) resulted in chronotropic and inotropic responses and a shortening of the interval between peak atrial and peak ventricular tensions (the A-V contraction interval). No dysrhythmias developed but at higher concentrations (590 micronM) 2 out of 7 hearts developed dysrhythmias (extrasystoles). 2. Perfusion with the antidepressants amitriptyline or maprotiline (4.8 micronM) or mianserin (28.8 micronM) reduced ventricular force, did not change heart rate and only amitriptyline reduced atrial force and lengthened the A-V contraction interval. At 4.8 micronM mianserin produced only a marginal shortening of the A-V contraction interval. 3. At these concentrations no dysrhythmias developed but at higher concentrations (amitriptyline 8 micronM, maprotiline 8 micronM, mianserin 60 micronM) all the agents produced dysrhythmias involving an interference with atrio-ventricular synchronization. 4. In the presence of mianserin (4.8 micronM) perfusion with noradrenaline (0.0059 to 5.9 micronM) shortened the A-V contraction interval and did not produce dysrhythmias. In the presence of amitriptyline or maprotiline (4.8 micronM) or mianserin (28.8 micronM) the A-V contraction interval generally lengthened and most hearts developed dysrhythmias (usually involving interference with atrio-ventricular synchronization). 5. [3H]-(-)-Noradrenaline uptake in perfused rabbit hearts and in mouse isolated atria or vasa deferentia was inhibited by the antidepressants to a similar extent, amitriptyline being marginally most potent (molar potency taken as 1.0), maprotiline being less potent (1.5) and mianserin least potent (2.0)). 6. It is concluded that of these three antidepressants, mianserin is least cardiotoxic in this preparation and that the ability of these antidepressants to predispose to noradrenaline-induced dysrhythmias is not related to blockade of noradrenaline uptake.