Effect of cholesterol re-supplementation and atorvastatin on plaque composition in the thoracic aorta of New Zealand white rabbits.

BACKGROUND: Effects of re-supplementation of a cholesterol-enriched diet (CEDrs) on size, cholesterol content and morphology of already existing plaques are not known to date. METHODS: A group of rabbits received standard chow (SC) for 6 weeks ("negative control"; for plasma lipid measurements only). Group I-IV received 2% CED (induction) for 6 weeks; thereafter, groups II-IV have been fed a SC (= cholesterol withdrawal) for 68 weeks. Afterwards, feeding of groups II-IV was continued as follows: Group II - 10 weeks SC, group III - 4 weeks 0.5% CED (~re-supplementation), afterwards 6 weeks SC (~withdrawal again); group IV - 4 weeks 0.5% CED (re-supplementation) + atorvastatin (2.5 mg/kg body weight/day), afterwards 6 weeks SC (~withdrawal again) + atorvastatin. Plasma lipids, but also plaque size, morphology and cholesterol contents of thoracic aortas were quantified. RESULTS: After CEDrs, plasma cholesterol levels were increased. However, after withdrawal of CEDrs, plasma cholesterol levels decreased, whereas the cholesterol content of the thoracic aorta was increased in comparison with the group without CEDrs. Plaque size remained unaffected. Atorvastatin application did not change plasma cholesterol level, cholesterol content of the thoracic aorta and plaque size in comparison with the group without drug treatment. However, atorvastatin treatment increased the density of macrophages (MΦ) compared with the group without treatment, with a significant correlation between densities of MΦ (Mac-1+) and apoptotic (TUNEL+; TP53+), antigen-presenting (HLA-DR+) or oxidatively stressed (SOD2+) cells. CONCLUSIONS: In rabbits with already existing plaques, CEDrs affects plaque morphology and cellular composition, but not plaque size. Despite missing effects on plasma cholesterol levels, cholesterol content of the thoracic aorta and size of already existing atherosclerotic plaques, atorvastatin treatment transforms the already existing lesions to a more active form, which may accelerate the remodelling to a more stable plaque.

Mass poisoning with NPS: 2C-E and Bromo-DragonFly.

BACKGROUND: Reports of intoxications with new psychoactive substances (NPS) mostly involve young people, as they are the main consumers of these types of drugs. This report centers on a case that was unusual due to it being a mass-poisoning event involving middle-aged individuals who had consumed a combination of the two different new psychoactive drugs 2,5-dimethoxy-4-ethylphenethylamine (2C-E) and 1-(8-bromofuro[2,3-f][1]benzofuran-4-yl)-2-propanamine (Bromo-DragonFly, BDF). CASE HISTORY: The mass poisoning of 29 individuals (24-56 years old) resulted in their admission to six different hospitals with severe symptoms of intoxication. All symptoms manifested after consumption of an unknown drug formulation around lunchtime during an esoteric weekend seminar. INVESTIGATION: Urine (n = 11) and blood samples (n = 29), collected from the 29 individuals for police investigation, were analyzed with immunochemical techniques, GC/MS and LC-MS/MS. 2C-E was confirmed in seven urine samples, but not in blood. BDF was confirmed in all urine samples, and in 17 blood samples. The blood samples exhibited BDF concentrations between ca. 0.6 and ca. 2.0 µg/L, while urine concentrations of BDF ranged from ca. 1.6 to 35 µg/L. The concentration of 2C-E in urine was found to be between ca. 1.5 and 183 µg/L. All patients made a complete recovery, although some had required mechanical ventilation. CONCLUSION: The investigation and the presentation of this case illustrates not only mass intoxication with 2C-E and BDF, with corresponding blood and urine concentrations, but also the necessity of collecting urine samples in cases where NPS-consumption is suspected, in order to improve the chances of analytical detection.

Correction to: Mass poisoning with NPS: 2C-E and Bromo-DragonFly.

The original version of this article contains an error. The Author S. Lehmann incorrectly listed as S. Lehman. The correct spelling is presented above. The original article has been corrected.

The fatty acid profile of the skin surface lipid layer in papulopustular rosacea.

BACKGROUND: Patients with papulopustular rosacea (PPR) frequently complain of dry, sensitive skin. We have previously demonstrated that patients with PPR have reduced skin surface hydration levels in the presence of normal sebum casual levels, suggesting that it may be the quality and not the quantity of sebum that plays a role in PPR. OBJECTIVES: To compare the sebaceous fatty acid composition of patients with PPR to that of controls with normal facial skin. METHODS: The sebaceous fatty acid composition of 25 patients with PPR and 24 age- and sex-matched controls was analysed by gas chromatography - mass spectrometry. Results Myristic acid (C14:0) was present in greater concentrations in PPR sebum, while the long chain saturated fatty acids arachidic acid (C20:0), behenic acid (C22:0), tricosanoic acid (C23:0) and lignoceric acid (C24:0) as well as the monounsaturated fatty acid cis-11-eicosanoic acid (C20:1) were present in the sebum of patients with PPR in lesser concentrations as compared with controls. CONCLUSIONS: There is increasing evidence that sebaceous fatty acids play a role in the maintenance of skin barrier integrity. We have shown for the first time that patients with PPR have an abnormal sebaceous fatty acid composition, with reduced levels of long chain saturated fatty acids. These new findings may have therapeutic implications for the development of sebum-modifying nonantibiotic treatments for patients with PPR.

Remodeling of inward rectifying K(+) currents in rat atrial myocytes by overexpression of A(1)-adenosine receptors.

In rat atrial myocytes GIRK (Kir3) channels can be activated by acetylcholine and adenosine via M(2) and A(1) receptors coupled to Pertussis-toxin-sensitive G proteins, such as M(2)R or A(1)R. Owing to the lower density of A(1)R, the amplitude of current activated by a saturating concentration (10 µM) of Ado (I(K(Ado))) amounts to about 40% of maximum I(K(ACh)). Adenovirus-driven overexpression of A(1)R results in an increase in I(K(Ado)). In a fraction of A(1)R-overexpressing cells, both ACh and Ado failed to activate GIRK channels. These cells had a large constitutive Ba(2+)-sensitive inward rectifying background K(+) current, which was insensitive to the GIRK channel inhibitor tertiapin (200 nM), suggesting this current component to be carried by I(K1) (Kir) channels. This effect of A(1)R overexpression was reduced by treatment (48 h) with the A(1)R antagonist DPCPX. siRNA-mediated knockdown of Kir2.1, simultaneously with A(1)R overexpression, substantially reduced I(K1). The mechanisms underlying the upregulation of functional I(K1) channels involve activation of the phosphatidylinositol 3-kinase (Pi3K)/Akt (protein kinase B) pathway. Kir2.1 transcripts are not increased in myocytes overexpressing A(1)R. These data demonstrate that manipulation of the expression level of a G protein-coupled receptor has unpredictable effects on functional expression of proteins that are supposed to be unrelated to the pathway controlled by that GPCR.

Metabolite concentrations in follicular fluid may explain differences in fertility between heifers and lactating cows.

There has been a marked decline in the fertility of dairy cows over the past decades, and metabolomic analysis offers a potential to investigate the underlying causes. Metabolite composition of the follicular fluid, which presents the intrafollicular environment, may be an important factor affecting oocyte maturation and subsequent early embryo development. The aim of the present study was to investigate the metabolic differences between follicular fluid from the dominant follicle of lactating cows and heifers using gas chromatography mass spectrometry (GC-MS)-based metabolomics. Follicular fluid and serum were collected from cows and heifers over three phases of follicle development: newly selected dominant follicles, preovulatory follicles prior to oestrus and post-LH surge follicles. Analysis of the fatty acids revealed that there were 24 fatty acids and 9 aqueous metabolites significantly different between cows and heifers. Of particular interest were the higher concentrations of saturated fatty acids (palmitic acid, P=0.001; stearic acid, P=0.005) in follicular fluid from cows and higher docosahexaenoic acid levels (P=0.022) in follicular fluid from heifers. Analysis of the metabolite composition of serum revealed that follicular fluid had a unique lipid composition. The higher concentrations of detrimental saturated fatty in cows will have a negative impact on oocyte maturation and early embryo development. Overall, the results suggest that the follicle microenvironment in cows potentially places their oocytes at a developmental disadvantage compared with heifers, and that this may contribute to well-characterised differences in fertility.

Wind streaks on venus: clues to atmospheric circulation.

Magellan images reveal surface features on Venus attributed to wind processes. Sand dunes, wind-sculpted hills, and more than 5830 wind streaks have been identified. The streaks serve as local "wind vanes," representing wind direction at the time of streak formation and allowing the first global mapping of near-surface wind patterns on Venus. Wind streaks are oriented both toward the equator and toward the west. When streaks associated with local transient events, such as impact cratering, are deleted, the westward component is mostly lost but the equatorward component remains. This pattern is consistent with a Hadley circulation of the lower atmosphere.

CTLA-4 CT60 polymorphism in thyroid and polyglandular autoimmunity.

Autoimmune thyroid diseases (AITDs) frequently occur together with other endocrine autoimmune conditions, denominated as polyglandular autoimmunity (PGA). The cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene was recently associated with AITD and PGA, and the CTLA-4 protein is a strong inhibitor of T-cells.The tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine. This study aimed to analyze the association of the CTLA-4 CT60 and TNF-alpha-863 polymorphisms with PGA. Homogeneous groups of 70 patients with AITD, 70 with type 1 diabetes (T1D), 70 with both AITD and T1D (PGA), and 100 healthy controls were genotyped for the CTLA-4 CT60 and TNF-alpha-863 polymorphisms by minisequencing on an ABI PRISM-3100 genetic analyzer. The CT60 G/G genotype was significantly more common in patients with PGA than in healthy controls (48.6 % vs. 32.0 % , OR = 2.01, 95 % CI = 1.07-3.77, p = 0.038). The CT60 allele frequencies differed as well between PGA patients and controls, with the predisposing G allele being increased in PGA (OR = 1.63, 95 % CI = 1.03-2.55, p = 0.042). Patients with PGA did not differ from those with AITD (p = 0.602) or T1D(p = 0.362). For TNF-alpha-863, carriers of the minor A allele occurred more frequently in the T1D group than in controls (47.1 % vs. 33 % , OR = 1.81, 95 % CI = 0.97-3.39, p = 0.079), but no differences in allele or genotype distribution were noted between PGA patients and controls (p = 0.886 and 0.389, respectively). In conclusion the CTLA-4 CT60 polymorphism is associated with PGA.

Postnatal development of cannabinoid receptor type 1 expression in rodent somatosensory cortex.

Endocannabinoids are powerful modulators of synaptic transmission that act on presynaptic cannabinoid receptors. Cannabinoid receptor type 1 (CB1) is the dominant receptor in the CNS, and is present in many brain regions, including sensory cortex. To investigate the potential role of CB1 receptors in cortical development, we examined the developmental expression of CB1 in rodent primary somatosensory (barrel) cortex, using immunohistochemistry with a CB1-specific antibody. We found that before postnatal day (P) 6, CB1 receptor staining was present exclusively in the cortical white matter, and that CB1 staining appeared in the gray matter between P6 and P20 in a specific laminar pattern. CB1 staining was confined to axons, and was most prominent in cortical layers 2/3, 5a, and 6. CB1 null (-/-) mice showed altered anatomical barrel maps in layer 4, with enlarged inter-barrel septa, but normal barrel size. These results indicate that CB1 receptors are present in early postnatal development and influence development of sensory maps.

The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents.

Monofunctional alkylating agents like methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are potent inducers of cellular stress leading to chromosomal aberrations, point mutations, and cell killing. We show that these agents induce a specific cellular stress response program which includes the activation of Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), p38 mitogen-activated protein kinase, and the upstream kinase SEK1/MKK4 and which depends on the reaction mechanism of the alkylating agent in question. Similar to another inducer of cellular stress, UV irradiation, damage of nuclear DNA by alkylation is not involved in the MMS-induced response. However, in contrast to UV and other inducers of the JNK/SAPKs and p38 pathways, activation of growth factor and G-protein-coupled receptors does not play a role in the MMS response. We identified the intracellular glutathione (GSH) level as critical for JNK/SAPK activation by MMS: enhancing the GSH level by pretreatment of the cells with GSH or N-acetylcysteine inhibits, whereas depletion of the cellular GSH pool causes hyperinduction of JNK/SAPK activity by MMS. In light of the JNK/SAPK-dependent induction of c-jun and c-fos transcription, and the Jun/Fos-induced transcription of xenobiotic-metabolizing enzymes, these data provide a potential critical role of JNK/SAPK and p38 in the induction of a cellular defense program against cytotoxic xenobiotics such as MMS.

UV-Induced stabilization of c-fos and other short-lived mRNAs.

Irradiation of cells with short-wavelength ultraviolet light (UVC) changes the program of gene expression, in part within less than 15 min. As one of the immediate-early genes in response to UV, expression of the oncogene c-fos is upregulated. This immediate induction is regulated at the transcriptional level and is transient in character, due to the autocatalyzed shutoff of transcription and the rapid turnover of c-fos mRNA. In an experiment analyzing the kinetics of c-fos mRNA expression in murine fibroblasts irradiated with UVC, we found that, in addition to the initial transient induction, c-fos mRNA accumulated in a second wave starting at 4 to 5 h after irradiation, reaching a maximum at 8 h, and persisting for several more hours. It was accompanied by an increase in Fos protein synthesis. The second peak of c-fos RNA was caused by an UV dose-dependent increase in mRNA half-life from about 10 to 60 min. With similar kinetics, the mRNAs of other UV target genes (i.e., the Kin17 gene, c-jun, IkappaB, and c-myc) were stabilized (e.g., Kin17 RNA from 80 min to more than 8 h). The delayed response was not due to autocrine cytokine secretion with subsequent autostimulation of the secreting cells or to UV-induced growth factor receptor activation. Cells unable to repair UVC-induced DNA damage responded to lower doses of UVC with an even greater accumulation of c-fos and Kin17 mRNAs than repair-proficient wild-type cells, suggesting that a process in which a repair protein is involved regulates mRNA stability. Although resembling the induction of p53, a DNA damage-dependent increase in p53 was not a necessary intermediate in the stabilization reaction, since cells derived from p53 knockout mice showed the same pattern of c-fos and Kin17 mRNA accumulation as wild-type cells. The data indicate that the signal flow induced by UV radiation addresses not only protein stability (p53) and transcription but also RNA stability, a hitherto-unrecognized level of UV-induced regulation.

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.

Photoproducts in transcriptionally active DNA induce signal transduction to the delayed U.V.-responsive genes for collagenase and metallothionein.

Mammalian cells in culture react to ultraviolet irradiation with the massive transcriptional activation of several genes and with the stabilization of the p53 protein. While U.V.-induced transcription of several immediate-response genes depends on U.V.-induced activation of signal transduction generated by non-nuclear mechanisms, stabilization of p53 and the transcription of several delayed-response genes are triggered by U.V.-induced DNA damage. By comparing dose responses for the activation by U.V. of delayed-responsive genes (collagenase 1, metallothionein IIA) in cells from patients with different DNA repair deficiencies (complementation groups of Xeroderma pigmentosum, Cockayne's syndrome and Trichothiodystrophy), we show here that U.V.-induced transcription of these genes does depend on pyrimidine dimers in transcribed regions of the genome (but not on damage in its silent part). Since all cells with defects in DNA repair that had been tested and which lack different enzymes, respond to U.V. with expression of these same genes, functional repair does not appear to be required for the induction of expression, and repair intermediates (which would not be identical in cells of different repair deficiency) cannot be responsible for signal generation.

Recombinant human O6-alkylguanine-DNA alkyltransferase induces conformational change in bound DNA.

Circular dichroism, and steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT bound to ds-DNA. Contrary to fluorescence, analysis of the far-UV CD spectra indicated a conformational change of AGT upon binding to DNA: its alpha-helical content is increased by approximately 12%. Analysis of near-UV CD spectra revealed that DNA was also affected, probably being separated into single strands locally.

Activation of muscarinic K+ current by beta-adrenergic receptors in cultured atrial myocytes transfected with beta1 subunit of heterotrimeric G proteins.

Muscarinic K+ channels (IK(ACh)) in native atrial myocytes are activated by betagamma subunits of pertussis toxin (Ptx)-sensitive heterotrimeric G proteins coupled to different receptors. betagamma subunits of Ptx-insensitive Gs, coupled to beta-adrenergic receptors, do not activate native IK(ACh). In atrial myocytes from adult rats transfected with rat brain beta1 subunit IK(ACh) can be activated by stimulation of beta-adrenergic receptors using isoprenaline. This effect is insensitive to Ptx. These findings demonstrate for the first time promiscuous (Ptx-insensitive) coupling of Gsbetagamma to GIRK channels in their native environment.

Antisense oligonucleotides against receptor kinase GRK2 disrupt target selectivity of beta-adrenergic receptors in atrial myocytes.

K+ channels composed of GIRK subunits are predominantly expressed in the heart and various regions of the brain. They are activated by betagamma-subunits released from pertussis toxin-sensitive G-proteins coupled to different seven-helix receptors. In rat atrial myocytes, activation of K(ACh) channels is strictly limited to receptors coupled to pertussis toxin-sensitive G-proteins. Upon treatment of myocytes with antisense oligodesoxynucleotides against GRK2, a receptor kinase with Gbetagamma binding sites, in a fraction of cells, K(ACh) channels can be activated by beta-adrenergic receptors. Sensitivity to beta-agonist is insensitive to pertussis toxin treatment. These findings demonstrate a potential role of Gbetagamma binding proteins for target selectivity of G-protein-coupled receptors.

Instability of the nuclear chromatin of procyclic Trypanosoma brucei brucei.

Digestion of nuclear chromatin of Trypanosoma brucei brucei procyclic culture forms with micrococcal nuclease yielded DNA fragments which formed DNA ladders in agarose gels, similar to those of rat liver. However, the chromatin of trypanosomes was digested more rapidly. The digestion of T. b. brucei chromatin yielded a large amount of DNA fragments of core-particle size. The numbers of base pairs per nucleosomal and linker DNA were identical in both species, if the digestion conditions were reduced in the case of T. b. brucei. Psoralen cross-linking of soluble chromatin of trypanosomes at 5 mM salt at pH 7 or pH 10 resulted in an irregular array of single-stranded (ss) bubbles separated by variable stretches of double-stranded (ds) DNA. The proportion of ss DNA was low compared with the ratio of ss/ds stretches in rat liver chromatin, which also showed regularly arranged nucleosomal DNA. Soluble chromatin of T. b. brucei, pre-treated with 500 mM NaCl to remove a potential H1 and psoralen cross-linked at 5 mM salt at pH 7 or pH 10 was to a great extent ds in both situations. The true nucleosome filament organization of T. b. brucei chromatin could only be shown by psoralen cross-linking the DNA in whole nuclei under physiological conditions. The results indicate that the chromatin of procyclic T. b. brucei differs significantly in its compaction pattern from rat liver chromatin; a typical histone H1 is not found, and the DNA-protein interactions are also less stable and can more easily be destabilized by experimental conditions.

The chromatin of trypanosomes.

The nuclear chromatin of trypanosomes is organised in the form of nucleosome filaments. When soluble chromatin is prepared under suitable conditions, a regular array of nucleosomes can be shown by electron microscopy. Chromatin of blood stream as well as procyclic culture forms of Trypanosoma brucei brucei and of T. cruzi shows limited compaction at salt concentrations increasing from 1 to 100 mM. No 30 nm fibres, typical for higher eukaryotes, are formed. Digestion of the nuclear chromatin with micrococcal nuclease and analysis of the histone proteins with various techniques reveal that the basic organisation of the trypanosome chromatin is similar but not identical as compared to that of higher eukaryotes. Distinct differences are present with respect to biochemical properties of the histones as well as to their interaction with the DNA. The primary structure of the histones also differs significantly from that found in other lower and higher eukaryotes. The function of the recently described H1-like proteins in trypanosomes is currently being investigated. The differences that have already been found in the structure and compaction of the trypanosome chromatin compared to that of higher eukaryotes lead us to expect differences of gene expression which, in turn, might offer targets for the control of trypanosomiasis.

Schizophrenia and birth order in Pakistan.

The paper describes a two stage study. In Stage I the birth orders of 453 adult patients with different diagnoses, seen in the routine work of a general hospital psychiatry department, were compared. Patients with schizophrenia had significantly higher average birth positions than patients with other diagnoses, even after controlling for sibship size.In Stage II, the birth positions of 64 patients with schizophrenia (DSMIIIR) were subjected to a goodness-of-fit chi-square test. Over-representation of eldest siblings was highly significant for both sexes. When patients aged 30 and above were analysed separately, there was still a significant excess of first-born. These findings are in contrast to birth order studies of schizophrenia in western populations. In the authors' opinion, they can not be accounted for by the biases to which birth order studies are prone. They indicate a need for further community-based studies.

Structural differences between the chromatin of procyclic Trypanosoma brucei brucei and of higher eukaryotes as probed by immobilized trypsin.

Soluble chromatin of Trypanosoma brucei brucei procyclic culture forms was submitted to digestion with free or immobilized trypsin. Digestion with trypsin in salt solutions of low and high ionic strengths generated characteristic sets of limit histone peptides. After incubation of chromatin with immobilized trypsin in a solution of low ionic strength, histones were not degraded, whereas a selective proteolysis occurred at 50 mM NaCl. Histones a and d, which correspond to H3 and H4 of higher eukaryotes, were rapidly attacked. Histones b and c, the counterparts of H2A and H2B, were more resistant. The results indicated that probably the basic N-terminal tails of the proteins a and d are located on the surface of the core particle. The location of d on the surface differs from the internal one proposed for histone H4. The salt-induced increase of susceptibility of histones to proteolysis reflects structural changes of T.b. brucei chromatin, which may result in partial chromatin compaction.