Anti-apolipoprotein A-1 autoantibodies as biomarker for atherosclerosis burden in patients with periodontitis.

BACKGROUND AND OBJECTIVE: Anti-apolipoprotein A-1 (anti-apoA-1) IgG is a potential marker of atherosclerotic plaque vulnerability and cardiovascular complications. In patients with periodontitis the presence of anti-apoA-1 IgGs in serum and their association with atherosclerosis is unknown. MATERIAL AND METHODS: One-hundred and thirty subjects with periodontal disease and 46 healthy subjects, matched for age and gender, participated in this study. Anti-apoA-1 IgG, high-sensitivity C-reactive protein (hsCRP) and matrix metalloproteinase (MMP) -2, -3, -8 and -9 were measured in serum samples. An ankle-brachial index (ABI) value below 1.11 served as a surrogate marker of atherosclerosis. Predictive accuracies of biomarkers for abnormal ABI were determined using receiver-operating characteristics curves and logistic regression analyses. RESULTS: Compared with healthy controls, periodontitis patients showed lower median ABI values (1.10 vs. 1.15; p < 0.0001), a higher prevalence of anti-apoA-1 IgG positivity (23.8% vs. 6.5%; p = 0.009) and higher concentrations of hsCRP (1.62 mg/L vs. 0.85 mg/L; p = 0.02) and MMP-9 (435 µg/mL vs. 283 µg/mL; p < 0.0001). In patients younger than 50 years of age (n = 66), anti-apoA-1 IgG was found to be the best predictor for an abnormal ABI (area under the curve = 0.63; p = 0.03). Anti-apoA-1 IgG positivity increased the risk of having an abnormal ABI (odds ratio = 4.20; p = 0.04), independently of diabetes, smoking and body mass index. CONCLUSIONS: Anti-apoA-1 IgG positivity and atherosclerosis, as reflected by abnormal ABI, were more prevalent in periodontitis patients than in age- and gender-matched controls. In younger periodontitis patients, anti-apoA-1 IgG was found to be the best predictor of atherosclerosis burden.

Transcriptomic profiling reveals differential cellular response to copper oxide nanoparticles and polystyrene nanoplastics in perfused human placenta.

The growing nanoparticulate pollution (e.g. engineered nanoparticles (NPs) or nanoplastics) has been shown to pose potential threats to human health. In particular, sensitive populations such as pregnant women and their unborn children need to be protected from harmful environmental exposures. However, developmental toxicity from prenatal exposure to pollution particles is not yet well studied despite evidence of particle accumulation in human placenta. Our study aimed to investigate how copper oxide NPs (CuO NPs; 10-20 nm) and polystyrene nanoplastics (PS NPs; 70 nm) impact on gene expression in ex vivo perfused human placental tissue. Whole genome microarray analysis revealed changes in global gene expression profile after 6 h of perfusion with sub-cytotoxic concentrations of CuO (10 µg/mL) and PS NPs (25 µg/mL). Pathway and gene ontology enrichment analysis of the differentially expressed genes suggested that CuO and PS NPs trigger distinct cellular response in placental tissue. While CuO NPs induced pathways related to angiogenesis, protein misfolding and heat shock responses, PS NPs affected the expression of genes related to inflammation and iron homeostasis. The observed effects on protein misfolding, cytokine signaling, and hormones were corroborated by western blot (accumulation of polyubiquitinated proteins) or qPCR analysis. Overall, the results of the present study revealed extensive and material-specific interference of CuO and PS NPs with placental gene expression from a single short-term exposure which deserves increasing attention. In addition, the placenta, which is often neglected in developmental toxicity studies, should be a key focus in the future safety assessment of NPs in pregnancy.

Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis.

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.

Elements involved in oxygen regulation of the Saccharomyces cerevisiae CYC7 gene.

The CYC7 gene of Saccharomyces cerevisiae encodes the minor species, iso-2, of the cytochrome c protein. Its expression is governed by two regulatory sequences upstream from the gene: a positive site which stimulates transcription 240 base pairs 5' from the protein-coding sequence (-240) and a negative site which inhibits transcription at -300. In this study, the nature of the positive site and its relationship to the negative site has been investigated. Expression of the CYC7 gene is weakly inducible by oxygen. This effect was greatly enhanced by the semidominant CYP1-16 mutation in the trans-acting gene CYP1. The weak oxygen regulation in wild-type cells and the enhanced induction in CYP1-16 mutants were found to be mediated through the positive site. A mutational analysis of this site implicated at least part of a tandem, direct repeat of 9 base pairs as essential for the functioning of this site. The relationship between the positive and negative sites was investigated by comparing the expression of the intact gene with that of derivatives lacking either one or the other site. The expression of the gene containing only the negative site was actually stimulated anaerobically, while the gene containing the positive site alone, although having higher expression aerobically than anaerobically, had higher anaerobic expression than did the intact gene. Thus, it appeared that the combination of the positive and negative sites suppressed anaerobic expression. A model which attempts to explain these properties of the two sites and account for the regulation of the expression of the intact gene is presented.

Interaction of graphene-related materials with human intestinal cells: an in vitro approach.

Graphene-related materials (GRM) inherit unique combinations of physicochemical properties which offer a high potential for technological as well as biomedical applications. It is not clear which physicochemical properties are the most relevant factors influencing the behavior of GRM in complex biological environments. In this study we have focused on the interaction of GRM, especially graphene oxide (GO), and Caco-2 cells in vitro. We mimiked stomach transition by acid-treatment of two representative GRM followed by analysis of their physicochemical properties. No significant changes in the material properties or cell viability of exposed Caco-2 cells in respect to untreated GRM could be detected. Furthermore, we explored the interaction of four different GO and Caco-2 cells to identify relevant physicochemical properties for the establishment of a material property-biological response relationship. Despite close interaction with the cell surface and the formation of reactive oxygen species (ROS), no acute toxicity was found for any of the applied GO (concentration range 0-80 µg ml(-1)) after 24 h and 48 h exposure. Graphene nanoplatelet aggregates led to low acute toxicity at high concentrations, indicating that aggregation, the number of layers or the C/O ratio have a more pronounced effect on the cell viability than the lateral size alone.

Type I oculocutaneous albinism associated with a full-length deletion of the tyrosinase gene.

Type I oculocutaneous albinism is an autosomal recessive disorder in which the biosynthesis of melanin is reduced or absent in skin, hair, and eyes because of deficient activity of tyrosinase (EC 1.14.18.1). Type I oculocutaneous albinism is caused by mutations in the tyrosinase structural gene, TYR; however, no large TYR gene deletions have been identified previously in humans. Here we report a patient with type IB oculocutaneous albinism who is a compound heterozygote for TYR allele containing a mutation that is likely to affect pre-RNA splicing and a paternally inherited allele in which the TYR gene is completely deleted, the first such allele described to date. Aside from the albinism in the proband, his phenotype and that of his normally pigmented father is otherwise normal, suggesting that this TYR deletion does not involve other functionally important contiguous genes.

Punctate appearance of dopamine-beta-hydroxylase on the chromaffin cell surface reflects the fusion of individual chromaffin granules upon exocytosis.

A secretion from cultured bovine chromaffin cells was stimulated to examine the pattern of exocytotic fusion on the plasma membrane. Confocal microscopy revealed that dopamine-beta-hydroxylase immunofluorescence in intact cells stimulated for 20s with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium was almost entirely punctate and evenly distributed on the cell surface. The basis for the fine, punctate appearance of dopamine-beta-hydroxylase was investigated. Dopamine-beta-hydroxylase presentation on the surface of permeabilized cells stimulated with 1-30 microM Ca2+ was punctate and similar to that on the plasma membrane of intact cells. The fluorescence intensities of both surface dopamine-beta-hydroxylase sites and internal chromaffin granules were estimated by computerized digital image analysis. The surface area of punctate surface dopamine-beta-hydroxylase (0.218 +/- 0.013 microm2, mean +/- S.E.M.) is similar to the surface area of a 0.28 microm diameter chromaffin granule (0.25 microm2). The average fluorescence intensity integrated over the area of the surface spots was 25-30% of the average chromaffin granule intensity, a fraction that is similar to the published values of 40-50% of the dopamine-beta-hydroxylase in the chromaffin granule being membrane bound. The surface density of the spots is consistent with the number of granules undergoing exocytosis. The spots do not tend to be clumped. The key conclusions from this work are that each individual punctate site of dopamine-beta-hydroxylase represents the fusion of a single chromaffin granule and that the distribution of dopamine-beta-hydroxylase spots over the cell surface is extensive and random, suggesting that each individual granule associates with its own release site.

Dicopper peroxo complexes: low temperature stopped-flow reaction kinetics of [(BQPA)CuI]+ and [(Me2-TMPA)CuI]+.

In the last 20 years, the reaction of many different CuI complexes and with dioxygen has been described. There is quite a big variety in their coordination geometry and most of them have been characterized by X-ray crystallography. Beyond structural information, stopped-flow kinetics experiments have provided additional mechanistic insights. In the particular systems [(BQPA)CuI]+ and [(Me2-TMPA)CuI]+ a new equilibrium between the two species trans-mu-1,2-peroxo and bis-mu-oxo is demonstrated. In the case of [(BQPA)CuI]+ the two species are in an equilibrium, presumably via the transient superoxo species. The reaction of [(Me2-TMPA)CuI]+ with dioxygen leads to the parallel formation of both species. The kinetically preferred trans-mu-peroxo species is then isomerised to the thermodynamically more stable bis-mu-oxo species.

Carbon nanotubes - curse or blessing.

Although nanotechnology is a relatively new scientific field, quite many different products are already introduced in the market containing nanosized particles. A special class of nanosized materials namely the carbon nanotubes (CNT) possesses outstanding new properties and extraordinary potential for creating new products. Carbon nanotubes are already used in various consumer products, industrial applications and science. It is not as this time clear how CNT are able to affect human health since most types of CNTs differ significantly in terms of structural characteristics (morphology, size, shape and length), surface properties (surface chemistry and surface charge) and chemical composition. This review provides an overview about contradicting reports that are found in the literature. We summarize the studies that report about nontoxic as well as toxic effects of CNT in-vitro and in-vivo. We describe how carbon nanotubes can readily be degraded under certain conditions. Another phenomenon is that despite the observed toxic effects which may occur to cells, organs and animals after uptake of CNT, intensive research investigations were undertaken in order to use these outstanding materials in medical applications. The second part of this review starts with a short description of the main principles in metrology. Observed conflicts were discussed in CNT toxicity assays into terms of measurement science or metrology issues. It was demonstrated that any specification of a measurand is only valid within the given framework. This means that many of the published results are within their measurement framework correct, but there are no means to compare them outside this framework.

C60 fullerene: a powerful antioxidant or a damaging agent? The importance of an in-depth material characterization prior to toxicity assays.

Since the discovery of fullerenes in 1985, these carbon nanospheres have attracted attention regarding their physico/chemical properties. Despite little knowledge about their impact on the environment and human health, the production of fullerenes has already reached an industrial scale. However, the toxicity of C(60) is still controversially discussed. The aim of this study was to clarify the biological effects of tetrahydrofuran (THF) suspended C(60) fullerene in comparison to water stirred C(60) fullerene suspensions. Beyond that, we analyzed the effects on the Crustacea Daphnia magna an indicator for ecotoxicological effects and the human lung epithelial cell line A549 as a simplified model for the respiratory tract. We could demonstrate that water-soluble side products which were formed in THF nC(60) suspension were responsible for the observed acute toxic effects, whereas fullerenes themselves had no negative effect regardless of the preparative route on either A549 cell in vitro or D. magna in vivo.

Intragenic TaqI restriction fragment length polymorphism (RFLP) in CICN4, between the loci for X-linked ocular albinism (OA1) and microphthalmia with linear skin defects syndrome (MLS).

A TaqI RFLP was detected within the ClCN4 gene, which lies between the loci for OA1 and MLS. There were no observed recombinations between this RFLP and the OA1 mutation in three informative families. Thus, the marker will be useful for genetic counseling in OA1.

Effects of expression of a mouse brain L-type calcium channel alpha 1 subunit on secretion from bovine adrenal chromaffin cells.

Regulated exocytosis from bovine chromaffin cells is stimulated by the influx of Ca2+ through plasma membrane ion channels that are opened by nicotinic stimulation and/or depolarization. Recently, we developed a novel method that enabled us to investigate the function of a cloned Ca2+ channel type C alpha 1 subunit in forming channels that stimulate exocytosis. In the present study, we demonstrate by immunocytochemistry that bovine chromaffin cells normally express an epitope specific for the type C alpha 1 subunit. We investigated the effects of expression of additional class C alpha 1 subunits (mouse brain clone) on various aspects of secretory function in bovine chromaffin cells by measuring secretion of cotransfected human growth hormone (GH, a reporter for the regulated secretory pathway in the transfected cells). New channels were activated in response to depolarization by both elevated K+ and nicotinic cholinergic agonist. The new channels had their greatest effects when secretion was stimulated suboptimally. Secretion was enhanced only after the first 30 sec of stimulation, and the enhancement extended beyond 5 min of continuous stimulation. In contrast to the endogenous L-type Ca2+ channels, the latency was not decreased by the dihydropyridine L-type Ca2+ channel agonist, Bay K 8644. The findings suggest that (i) the Ca(2+)-sensitive mechanism for triggering or maintaining exocytosis is capable of being saturated by high levels of Ca2+, (ii) secretion caused by nicotinic agonist stimulation can be significantly enhanced by activation of voltage-sensitive Ca2+ channels, and (iii) the effects on secretion of the L-type Ca2+ channels formed on expression of the mouse brain class C alpha 1 subunit are distinct from those of endogenous L-type Ca2+ channels.

Deletion mapping and a highly reduced radiation hybrid in the Xp22.3-p22.2 region.

The human Xp22.3-p22.2 region contains several known disease genes as well as distinctive families of low copy repetitive sequences. In this study, we have developed new tools to more finely map this area. We have characterized a mapping panel of various cell lines and hybrids with different molecular breakpoints as defined by previously mapped reference markers from this region. The panel subdivides this area into nine distinct regions from DXS41 through the pseudoautosomal boundary. We have also identified a radiation-reduced somatic cell hybrid, Z4-7, that contains DXS31, DXS452, STS, DXS143, and DXS85, but not PABX, DXS16, or other single-copy probes from proximal Xp and Xq. A phage library was constructed from Z4-7 and over 80,000 plaques were screened with total human DNA. More than 100 positive clones were identified as potential new markers in this region. Nine of these have been mapped to the hybrid panel, and unique subclones have been isolated from three of these markers. The panel has also allowed us to map several other DNA markers, genes (AMG, OA1), and repetitive elements of the DXF22S and DXF30S sequence families relative to the various breakpoints.

Fluorescence resolution of the intrinsic tryptophan residues of bovine protein tyrosyl phosphatase.

Fluorescence steady-state and lifetime measurements have been performed that permit the differentiation of the 2 intrinsic tryptophan residues in bovine low molecular weight phosphotyrosyl protein phosphatase (BPTP). Spectral information was obtained by use of two single-tryptophan mutant proteins, W39F and W49F, and the double mutant protein W39,49F. Fluorescence measurements show that Trp39 is characterized by a large blue shift, a low quantum yield, and a shorter mean lifetime compared to Trp49. Solute fluorescence quenching studies of W39F reveal that Trp49 is highly exposed to the aqueous environment. In contrast, Trp39 is situated within a hydrophobic core and is only partially accessible to quenching agents such as acrylamide, iodide ion, and cesium ion. The fluorescence contributions of Trp39 and Trp49 are additive, and their sum is equivalent to that observed for wild type BPTP. Calculated intramolecular distances between Trp39 or Trp49 and a 5-[[(acetylamino)-ethyl]amino]naphthalene-1- sulfonate group covalently bound at Cys12 or Cys17 of the respective protein mutants, place Trp49 within 10 A and Trp39 at least 20 A from the active site. The fluorescence decay of the single tryptophan mutants and, surprisingly, wild type BPTP were each adequately fitted as biexponentials. The latter is a consequence of the imprecision involved in determining actual minima in a three- and four-exponential fitting. Comparison of quenching results of wild type BPTP with those of the single tryptophan mutant proteins indicates that minor fluorescence components, easily resolved using a biexponential fitting for the mutant proteins, are unresolvable for wild type BPTP. These minor components skewed the weighted magnitudes and induced perturbations in lifetimes for the tryptophan fluorescence of wild type BPTP, which directly influenced the calculated values of Ksv and kq.

Alpha-1 adrenergic coupling events induced by full and partial agonists in rabbit aorta.

Differences in the ability of full vs. partial agonists to initiate alpha-1 adrenergic receptor-mediated coupling events were studied in isolated segments of rabbit aorta. Mono- and dimethoxysubstituted tolazolines produced contractile responses which, at their maximum, were 27 to 100% of the response produced by the full agonist phenylephrine. In addition to differences in maximum response, contraction kinetics varied between full and partial agonists. Responses to partial agonists displayed a slower approach to peak tension and loss of the rapid phase of tension development which is associated with release of intracellular Ca++. Among the tolazoline series 3,5 dimethoxy-, 3 methoxy-, and 2 methoxy derivatives were compared further with phenylephrine for their ability to cause phosphatidylinositol cycle turnover, intracellular Ca++ release and Ca++ influx. For each of these coupling events, a rank of phenylephrine greater than or equal to 3, 5 greater than 3 greater than 2 was observed. However, a higher percentage of Ca++ influx vs. Ca++ release was observed for the partial agonists, suggesting that their contractile responses may be more dependent upon extracellular Ca++ than intracellular Ca++. Our results indicate that partial agonists initiate the same coupling events as full agonists; however, the relative proportion of Ca++ release and influx may be different for partial agonists because of the reduced rate of second messenger production.

Identification of Yersinia spp. with the API 20E system.

The ability of the API 20E system to identify 105 clinical isolates of Yersinia spp. was compared with those of conventional biochemical tests at 28 and 37 degrees C. Elimination of the Voges-Proskauer test (recorded as a negative result) increased the percentage of correct identifications for Yersinia spp. from 66 to 93% when the API 20E strips were incubated at 28 degrees C.

Transient transfection studies of secretion in bovine chromaffin cells and PC12 cells. Generation of kainate-sensitive chromaffin cells.

We have developed a transient transfection method to measure protein secretion from non-dividing, primary bovine chromaffin cells and from the continuous cell line, PC12. A plasmid coding human growth hormone (GH) was expressed in sufficient amounts in bovine chromaffin and PC12 cells to allow precise measurements of secretion from the small fraction (less than 1%) of transfected cells in a dish. GH was secreted in a similar proportion to endogenous catecholamine upon nicotinic stimulation, depolarization with elevated K+, and upon permeabilization with digitonin and subsequent stimulation with micromolar Ca2+. GH in homogenates from GH-transfected chromaffin cells cosedimented with catecholamine on discontinuous sucrose gradients. The data indicate that transiently expressed human GH in chromaffin and PC12 cells is localized predominantly in secretory vesicles in the regulated secretory pathway. With transient transfection there is a high probability of coexpression in the same cell of two plasmids which are cotransfected. Coexpression of a plasmid for GH and a plasmid for the non-N-methyl-D-aspartate glutamate receptor, GluR1, created chromaffin cells in which Ca(2+)-dependent GH secretion could be stimulated by the glutamatergic agonist kainate. The ability to coexpress a plasmid of interest with a plasmid for GH will allow the investigation of the role of other cloned proteins in the regulated secretory pathway in differentiated, non-dividing cells.

In-vitro studies of antibiotic combinations for multiply-resistant coagulase-negative staphylococci.

Coagulase-negative staphylococci are being implicated as pathogens with increasing frequency and this may in part be due to their development of resistance to a wide variety of antibiotics. Because vancomycin is the only drug generally available for treating these multiply resistant organisms, we have tested several combinations of antibiotics for activity in vitro against these organisms. These in-vitro studies suggested that rifampicin combined with either gentamicin or cefamandole might occasionally provide an efficacious alternative to vancomycin, while the novobiocin plus rifampicin combination might be an effective oral regimen against methicillin-resistant coagulase negative staphylococci. Resistance to co-trimoxazole was found in nearly all isolates tested.

Phenotypic variability in X-linked ocular albinism: relationship to linkage genotypes.

One hundred nineteen individuals from 11 families with X-linked ocular albinism (OA1) were studied with respect to both their clinical phenotypes and their linkage genotypes. In a four-generation Australian family, two affected males and an obligatory carrier lacked cutaneous melanin macroglobules (MMGs); ocular features were identical to those of Nettleship-Falls OA1. Four other families had more unusual phenotypic features in addition to OA1. All OA1 families were genotyped at DXS16, DXS85, DXS143, STS, and DXS452 and for a CA-repeat polymorphism at the Kallmann syndrome locus (KAL). Separate two-point linkage analyses were performed for the following: group A, six families with biopsy-proved MMGs in at least one affected male; group B, four families whose biopsy status was not known; and group C, OA-9 only (16 samples), the family without MMGs. At the set of loci closest to OA1, there is no clear evidence in our data set for locus heterogeneity between groups A and C or among the four other families with complex phenotypes. Combined multipoint analysis (LINKMAP) in the 11 families and analysis of individual recombination events confirms that the major locus for OA1 resides within the DXS85-DXS143 interval. We suggest that more detailed clinical evaluations of OA1 individuals and families should be performed for future correlation with specific mutations in candidate OA1 genes.

D4 dopamine receptor-mediated phospholipid methylation and its implications for mental illnesses such as schizophrenia.

Previous studies have shown D2-like dopamine receptor involvement in the regulation of phospholipid methylation (PLM), while others have documented impaired methionine and folate metabolism in schizophrenia. Utilizing [14C]formate labeling in cultured neuroblastoma cell lines, we now show that D4 dopamine receptors (D4R) mediate the stimulatory effect of dopamine (DA) on PLM. The effect of DA was potently blocked by highly D4R-selective antagonists and stimulated by the D4R-selective agonist CP-226269. DA-stimulated PLM was dependent upon the activity of methionine cycle enzymes, but DA failed to increase PLM in [3H]methionine labeling studies, indicating that a methionine residue in the D4R might be involved in mediating PLM. A direct role for MET313, located on transmembrane helix No. 6 immediately adjacent to phospholipid headgroups, was further suggested from adenosylation, site-directed mutagenesis and GTP-binding results. A comparison of PLM in lymphocytes from schizophrenia patients vs control samples showed a four-fold lower activity in the schizophrenia group. These findings reveal a novel mechanism by which the D4R can regulate membrane composition. Abnormalities in D4R-mediated PLM may be important in psychiatric illnesses such as schizophrenia.