Human saliva contains a large number of proteins that can be used for diagnosis and are of great potential in clinical and epidemiological research. The aim of this work was to map the proteins in saliva by two-dimensional gel electrophoresis (2-DE), and to identify abundant proteins by peptide mass fingerprinting using trypsin cleavage and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis. One hundred proteins were identified representing 20 different identities according to accession numbers. Abundant proteins expressed in different forms were: alpha-amylase, immunoglobulin A, prolactin-inducible protein, zinc-alpha(2)-glycoprotein and cystatins (S, SA, D and SN). Other proteins found were interleukin-1 receptor antagonist, von Ebner's gland protein (lipocalin-1) and calgranulin A and B (S100A8 and A9). Furthermore, apolipoprotein A-I, beta(2)-microglobulin, glutathione S-transferase P and fatty acid-binding protein were also identified. Our results show that human saliva contains a large number of proteins that are involved in inflammatory and immune responses. The 2-DE protein map constructed opens the possibility to investigate protein changes associated with disease processes.

OBJECTIVE

To investigate in adult rats the effects of two alpha(2)-selective adrenergic agonists (alpha(2)-SAs; AGN 191103 and AGN 190342) on retinal ganglion cell (RGC) survival after transient retinal ischemia.

METHODS

RGCs were labeled with a Fluorogold (FG) tracer applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 60 or 90 minutes. In one group, a single dose of saline or one alpha(2)-SA was administered intraperitoneally (IP) or topically 1 hour before ischemia. In another group, a single dose of AGN 190342 was administered IP, 1, 2, 4, 24, or 72 hours after ischemia. Rats were processed 7, 14, or 21 days later. Densities of surviving RGCs were estimated by counting FG-labeled cells in 12 standard retinal areas.

RESULTS

Seven days after 60 or 90 minutes of retinal ischemia, death had occurred in 36% or 47%, respectively, of the RGC population, and by 21 days the loss of RGCs amounted to 42% or 62%, respectively. Systemic pretreatment with an alpha(2)-SA resulted in enhanced survival of ischemic-injured RGCs. Topical pretreatment with an alpha(2)-SA prevented up to 100% of the ischemia-induced RGC loss. Pretreatment with an alpha(2)-SA abolished the secondary slow RGC loss that occurred between days 7 and 21 after ischemia. When administered shortly after ischemia (up to 2 hours) AGN 190342 rescued substantial proportions of RGCs destined to die and diminished slow RGC death.

CONCLUSIONS

Pretreatment and early posttreatment with an alpha(2)-SA induces marked long-lasting neuroprotective in vivo protection against ischemia-induced cell death in RGCs.

Female rats and mice have smaller and, per body mass (BM), more alveoli and alveolar surface area (Sa) than males of their respective species. This sexual dimorphism becomes apparent about the time of sexual maturity. It is prevented in rats (not tested in mice) by ovariectomy at age 3 wk. In female mice, estrogen receptor (ER)-alpha and ER-beta are required for formation of alveoli of appropriate size and number. We now report the average volume of an alveolus (va) and the number of alveoli per body mass (Na/BM) were not statistically different between ER-alpha(-/-) and wild type (wt) males. However, the combination of a larger value for va and a smaller value for Na/BM, though neither parameter achieved a statistically significant intergroup difference, resulted in a statistically significant lower Sa/BM in ER-alpha(-/-) males compared with wt males. In ER-beta(-/-) males, va was bigger and Na/BM and Sa/BM were lower compared with wt males. Wt males had larger alveoli and lower Na/BM and Sa/BM than wt females. The wt sexual dimorphism of va, Na/BM, and Sa/BM was absent in ER-alpha(-/-) mice. Alveolar size did not differ between ER-beta(-/-) females and males but Na/BM and Sa/BM were greater in ER-beta(-/-) females than in ER-beta(-/-) males. The results in male mice, with prior findings in female mice, 1) demonstrate estrogen receptors have a smaller effect on alveolar dimensions in male than female mice, 2) show ER-alpha and ER-beta are required for the sexual dimorphism of alveolar size, and 3) show ER-alpha is needed for the sexual dimorphism of body mass-specific alveolar number and surface area.

The dopamine (DA) mesolimbic pathway, which originates from DA cell bodies within the ventral tegmental area (VTA), has been shown by various studies to play a role in the mediation of various drugs of abuse including alcohol (EtOH). It has been suggested that the VTA's control of EtOH reward is mediated in part by the D2 receptors within the VTA. These receptors may be under the regulation of reciprocal GABAergic inputs from forebrain components of the mesolimbic path such as the nucleus accumbens (NAcc), a classic EtOH reward substrate, and the bed nucleus of the stria terminalis, a substrate recently implicated in EtOH reinforcement, forming a self-regulating feedback loop. To test this hypothesis, D2 regulation of EtOH self-administration (SA) was evaluated by the microinfusion of the D2 antagonist eticlopride into the VTA of P rats, which produced profound reductions in EtOH SA in the highest (20.0 and 40.0microg) doses tested in both BST/VTA and NAcc/VTA implanted P rats. To determine the role of GABA in the mediation of EtOH SA, a 32.0ng dose the non-selective GABA antagonist SR 95531 was microinfused into the BST producing no effect on responding for EtOH and into the NAcc which lead to a reduction in EtOH responding. Finally, the hypothesis that GABA innervation of the VTA from the mesolimbic forebrain may influence EtOH SA was examined by the simultaneous infusion of eticlopride (40.0microg) into the VTA and SR 95531 (32.0ng) into either the BST or NAcc. This combination infusion completely attenuated the reduction in EtOH SA observed with the 40.0microg dose of eticlopride alone in both groups of animals. These results suggest that while the D2 receptors within the VTA regulate EtOH-motivated behaviors, this is modulated by GABAergic input from the mesolimbic forebrain, specifically from the BST and NAcc.

BACKGROUND

The circulating estrogen concentration elevated gradually along with time after ovariectomy in rats. To explore the source of the increased circulation estrogen, the extragonadal aromatization as well as the synthesis of androgen in the adrenal cortex of the ovariectomized rats was evaluated.

METHODS

Female rats were divided into twelve groups: 1 month after ovariectomy (OVX1M), OVX2M, OVX3M, OVX4M, OVX5M, OVX6M; intact 1 month (INT1M), INT2M, INT3M, INT4M, INT5M, INT6M. The blood concentration of testosterone (T) was measured by radioimmunoassay. The mRNA expressions of P450 aromatase in the liver and subcutaneous abdominal (SA) adipose as well as the adrenal cytochrome P450 17 alpha hydroxylase/lyase (P450c17) were semiquantified by RT-PCR. The P450 aromatase protein expressions in the liver and SA adipose were detected by Western blot.

RESULTS

The blood E2 concentrations increased gradually along with time after ovariectomy in the rats. The 58-kDa aromatase protein and mRNA expressions normalized to beta-actin in the OVX6M rats' SA adipose tissues showed higher levels than those from corresponding tissues in the INT6M (p < 0.05). And the ratios of aromatase mRNA and protein to beta-actin in the OVX6M rats' liver tissues increased significantly compared with those in the OVX1M rats (p < 0.05). The ratio of adrenal P450c17 to beta-actin in the OVX6M increased markedly, and was higher than OVX1M (p < 0.05), though the blood concentration of T decreased significantly in all the ovariectomized rats (p < 0.05).

CONCLUSIONS

Both the subcutaneous abdominal adipose tissues and the liver tissues contributed to the extragonadal aromatisation to promote the circulating E2 levels in the rats along with time after ovariectomy; the adrenal compensation might also be activated naturally.

The Multidimensional Scale of Independent Functioning (MSIF) is a new instrument for rating functional disability in psychiatric outpatients. The MSIF differs from other disability rating scales by providing discrete ratings of (1) role responsibility, (2) presence and level of support, and (3) performance quality. The MSIF, which consists of a semistructured interview and detailed rating anchors, was validated in 114 psychiatric outpatients. The instrument had good criterion, discriminative, interrater, and construct validity. Correlations between comparable ratings on the Social Adjustment Scale II (SAS II) ranged from 0.78 to 0.86. Nevertheless, redundancy analysis using canonical correlation demonstrated that, although the two instruments overlap, the MSIF contains information that is not contained in the SAS II. Furthermore, there was only modest shared variance with conceptually non-overlapping subscales in the SAS II. Interrater reliability (intraclass correlation coefficients) ranged from 0.74 to 1.00 for global and subscale scores. MSIF subscales performed as expected with respect to external validators such as hours of employment, earned income, supported versus nonsupported employment and housing, and mainstream versus nonmainstream educational status. MSIF global ratings were modestly correlated with IQ and psychopathology ratings, consistent with reports in the literature. Construct validity, estimated using Cronbach's alpha coefficient, was 0.72. The MSIF is a promising new instrument designed to circumvent several limitations with existing functional outcome instruments for longitudinal studies, intervention research, and services research.

Fluctuation analysis experiments were performed in the human sarcoma cell line MES-SA to assess whether selection or induction mechanisms determine resistance to doxorubicin (DOX), mutation rates, and the nature of the surviving clones. Thirteen flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 3.3 x 10(6) cells. After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks. Surviving colonies were scored and harvested. Clones were propagated and analyzed for drug resistance phenotype. Expression of the mdr1, mrp, and topoisomerase II alpha and II beta genes was analyzed by reverse transcription-polymerase chain reaction. Accumulation of the P-glycoprotein substrate rhodamine-123 was measured by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833. Cellular glutathione levels were measured by flow cytometry, and M(r) 110,000 vesicular protein (p110) expression was detected by immunohistochemistry. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction conferring DOX resistance. At this stringent level (5-6 log cell killing) of drug exposure, the mutation rate was estimated at 1.8 x 10(-6) per cell generation. All 30 propagated clones demonstrated cross-resistance to vinblastine, etoposide, and paclitaxel (Taxol), but not to cisplatin or bleomycin. Increased mRNA levels of mdr1 were observed in all 27 clones tested, including at least 1 from each of the 13 populations. No alterations were found in expression or level of topoisomerase II alpha or II beta, mrp, glutathione, and p110. Expression of P-glycoprotein was confirmed by flow cytometry using the monoclonal antibody UIC2. In almost all tested clones, decreased intracellular rhodamine-123 accumulation was modulated by 2 microM SDZ PSC 833, and the vinblastine resistance in all examined clones was completely reversed by SDZ PSC 833 and verapamil. Our study demonstrates that survival of cells exposed to DOX in a single step occurs as a result of a stochastic process consistent with mutational events. Activation of the mdr1 gene is the predominant mechanism selected by DOX in these resistant clones.

Synaptopodin (SP) is a marker and essential component of the spine apparatus (SA), an enigmatic cellular organelle composed of stacked smooth endoplasmic reticulum that has been linked to synaptic plasticity. However, SP/SA-mediated synaptic plasticity remains incompletely understood. To study the role of SP/SA in homeostatic synaptic plasticity we here used denervation-induced synaptic scaling of mouse dentate granule cells as a model system. This form of plasticity is of considerable interest in the context of neurological diseases that are associated with the loss of neurons and subsequent denervation of connected brain regions. In entorhino-hippocampal slice cultures prepared from SP-deficient mice, which lack the SA, a compensatory increase in excitatory synaptic strength was not observed following partial deafferentation. In line with this finding, prolonged blockade of sodium channels with tetrodotoxin induced homeostatic synaptic scaling in wild-type, but not SP-deficient, slice cultures. By crossing SP-deficient mice with a newly generated transgenic mouse strain that expresses GFP-tagged SP under the control of the Thy1.2 promoter, the ability of dentate granule cells to form the SA and to homeostatically strengthen excitatory synapses was rescued. Interestingly, homeostatic synaptic strengthening was accompanied by a compensatory increase in SP cluster size/stability and SA stack number, suggesting that activity-dependent SP/SA remodeling could be part of a negative feedback mechanism that aims at adjusting the strength of excitatory synapses to persisting changes in network activity. Thus, our results disclose an important role for SP/SA in homeostatic synaptic plasticity.

In order to determine the efficacy of using polyanhydrides as a carrier for therapeutic proteins, the model protein bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC) was encapsulated in microspheres of poly sebacic anhydride (poly(SA)), and random copolymers of poly(SA) and poly(1,6-bis-p-carboxyphenoxy)hexane (poly(CPH)). The microspheres were fabricated via the double emulsion (water/oil/water) technique and were characterized using scanning electron microscopy, gel permeation chromatography, confocal microscopy, and a Coulter counter. The effect of protein loading, protein distribution, and change in polymer composition was examined in an in vitro release study. The secondary structure of the encapsulated BSA-FITC was determined with Fourier transform infrared spectroscopy. The primary structure of the released protein was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Poly(SA) and 20:80 (CPH:SA) microspheres were found to conserve the primary structure of the released protein and the secondary structure of the encapsulated protein, and showed a sustained delivery for approximately 15 and 30 days, respectively. As the CPH content in the copolymer increased, the secondary structure of FITC-BSA was not conserved, as indicated by the steep decrease in the alpha-helix content.

The spatial distribution of alpha- and beta-myosin heavy chain isoforms (MHCs) was investigated immunohistochemically in the embryonic human heart between the 4th and the 8th week of development. The development of the overall MHC isoform expression pattern can be outlined as follows: (1) In all stages examined, beta-MHC is the predominant isoform in the ventricles and outflow tract (OFT), while alpha-MHC is the main isoform in the atria. In addition, alpha-MHC is also expressed in the ventricles at stage 14 and in the OFT from stage 14 to stage 19. This expression pattern is very reminiscent of that found in chicken and rat. (2) In the early embryonic stages the entire atrioventricular canal (AVC) wall expresses alpha-MHC whereas only the lower part expresses beta-MHC. The separation of atria and ventricles by the fibrous annulus takes place at the ventricular margin of the AVC wall. Hence, the beta-MHC expressing part of the AVC wall, including the right atrioventricular ring bundle, is eventually incorporated in the atria. (3) In the late embryonic stages (approx. 8 weeks of development) areas of alpha-MHC reappear in the ventricular myocardium, in particular in the subendocardial region at the top of the interventricular septum. These coexpressing cells are topographically related to the developing ventricular conduction system. (4) In the sinoatrial junction of all hearts examined alpha- and beta-MHC coexpressing cells are observed. In the older stages these cells are characteristically localized at the periphery of the SA node.

Sulfated fucans are common structural components of the cell walls of marine brown algae. Using a fucan-degrading hydrolase isolated from a marine bacterium, we prepared sulfated fucan oligosaccharides made of mono- and disulfated fucose units alternatively bound by alpha-1,4 and alpha-1,3 glycosidic linkages, respectively. Here, we report on the elicitor activity of such fucan oligosaccharide preparations in tobacco. In suspension cell cultures, oligofucans at the dose of 200 microg ml(-1) rapidly induced a marked alkalinization of the extracellular medium and the release of hydrogen peroxide. This was followed within a few hours by a strong stimulation of phenylalanine ammonia-lyase and lipoxygenase activities. Tobacco leaves treated with oligofucans locally accumulated salicylic acid (SA) and the phytoalexin scopoletin and expressed several pathogenesis-related (PR) proteins, but they displayed no symptoms of cell death. Fucan oligosaccharides also induced the systemic accumulation of SA and the acidic PR protein PR-1, two markers of systemic acquired resistance (SAR). Consistently, fucan oligosaccharides strongly stimulated both local and systemic resistance to tobacco mosaic virus (TMV). The use of transgenic plants unable to accumulate SA indicated that, as in the SAR primed by TMV, SA is required for the establishment of oligofucan-induced resistance.

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.

The tumor development and metastasis are closely related to the structure and function of the tumor microenvironment (TME). Recently, TME modulation strategies have attracted much attention in cancer immunotherapy. Despite the preliminary success of immunotherapeutic agents, their therapeutic effects have been restricted by the limited retention time of drugs in TME. Compared with traditional delivery systems, nanoparticles with unique physical properties and elaborate design can efficiently penetrate TME and specifically deliver to the major components in TME. In this review, we briefly introduce the substitutes of TME including dendritic cells, macrophages, fibroblasts, tumor vasculature, tumor-draining lymph nodes and hypoxic state, then review various nanoparticles targeting these components and their applications in tumor therapy. In addition, nanoparticles could be combined with other therapies, including chemotherapy, radiotherapy, and photodynamic therapy, however, the nanoplatform delivery system may not be effective in all types of tumors due to the heterogeneity of different tumors and individuals. The changes of TME at various stages during tumor development are required to be further elucidated so that more individualized nanoplatforms could be designed.

Keywords: AC-NPs, antigen-capturing nanoparticles; ANG2, angiopoietin-2; APCs, antigen-presenting cells; Ab, antibodies; Ag, antigen; AuNCs, gold nanocages; AuNPs, gold nanoparticles; BBB, blood-brain barrier; BTK, Bruton's tyrosine kinase; Bcl-2, B-cell lymphoma 2; CAFs, cancer associated fibroblasts; CAP, cleavable amphiphilic peptide; CAR-T, Chimeric antigen receptor-modified T-cell therapy; CCL, chemoattractant chemokines ligand; CTL, cytotoxic T lymphocytes; CTLA4, cytotoxic lymphocyte antigen 4; CaCO3, calcium carbonate; Cancer immunotherapy; DCs, dendritic cells; DMMA, 2,3-dimethylmaleic anhydrid; DMXAA, 5,6-dimethylxanthenone-4-acetic acid; DSF/Cu, disulfiram/copper; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; EPG, egg phosphatidylglycerol; EPR, enhanced permeability and retention; FAP, fibroblast activation protein; FDA, the Food and Drug Administration; HA, hyaluronic acid; HB-GFs, heparin-binding growth factors; HIF, hypoxia-inducible factor; HPMA, N-(2-hydroxypropyl) methacrylamide; HSA, human serum albumin; Hypoxia; IBR, Ibrutinib; IFN-γ, interferon-γ; IFP, interstitial fluid pressure; IL, interleukin; LMWH, low molecular weight heparin; LPS, lipopolysaccharide; M2NP, M2-like TAM dual-targeting nanoparticle; MCMC, mannosylated carboxymethyl chitosan; MDSCs, myeloid-derived suppressor cells; MPs, microparticles; MnO2, manganese dioxide; NF-κB, nuclear factor κB; NK, nature killer; NO, nitric oxide; NPs, nanoparticles; Nanoparticles; ODN, oligodeoxynucleotides; PD-1, programmed cell death protein 1; PDT, photodynamic therapy; PFC, perfluorocarbon; PHDs, prolyl hydroxylases; PLGA, poly(lactic-co-glycolic acid); PS, photosensitizer; PSCs, pancreatic stellate cells; PTX, paclitaxel; RBC, red-blood-cell; RLX, relaxin-2; ROS, reactive oxygen species; SA, sialic acid; SPARC, secreted protein acidic and rich in cysteine; TAAs, tumor-associated antigens; TAMs, tumor-associated macrophages; TDPA, tumor-derived protein antigens; TGF-β, transforming growth factor β; TIE2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2; TIM-3, T cell immunoglobulin domain and mucin domain-3; TLR, Toll-like receptor; TME, tumor microenvironment; TNF-α, tumor necrosis factor alpha; TfR, transferrin receptor; Tregs, regulatory T cells; Tumor microenvironment; UPS-NP, ultra-pH-sensitive nanoparticle; VDA, vasculature disrupting agent; VEGF, vascular endothelial growth factor; cDCs, conventional dendritic cells; melittin-NP, melittin-lipid nanoparticle; nMOFs, nanoscale metal-organic frameworks; scFv, single-chain variable fragment; siRNA, small interfering RNA; tdLNs, tumor-draining lymph nodes; α-SMA, alpha-smooth muscle actin.

OBJECTIVE

An increased sympathetic drive, in view of its proarrhythmic, proatherosclerotic, and prothrombotic actions, could contribute to the elevated cardiovascular risk of habitual smokers. However, the underlying mechanisms are still debated. In this study we address the hypothesis that spectral analysis of RR interval and systolic arterial pressure short-term variabilities may be used to assess the complex autonomic changes produced by habitual cigarette smoking.

METHODS

A cross-sectional design compared heavy >> 20 cigarettes/day) habitual smokers (n = 20; 40 +/- 3 years), with similar age controls. Spectral analysis of RR interval variability provided markers of the sympatho-vagal balance modulating the SA node, by way of the normalised low frequency (LF approximately equal to 0.10 Hz) and high frequency (HF approximately equal to 0.25 Hz) components. The LF component of systolic arterial pressure (SAP) variability assessed the sympathetic vasomotor modulation. The frequency domain index (alpha) measured the baroreflex gain of the SA node. Subjects were studied at rest, and during the sympathetic excitation produced by active standing.

RESULTS

In smokers LFRR was, at rest, greater than in controls (70.6 +/- 3.8 vs 46.0 +/- 2.5 normalised units, nu); concurrently HFRR was reduced (22.1 +/- 3.2 vs 42.0 +/- 2.8 nu). Baroreflex gain and RR variance were also smaller in smokers. LFSAP was, instead, similar in the smokers and control groups. The standing induced increase in LFRR was blunted (P < 0.001) in smokers.

CONCLUSIONS

Spectral analysis of RR interval and systolic arterial pressure variability indicates that habitual cigarette smoking induces selective alterations in neural control of the SA node. An increase at rest in markers of sympathetic modulation is accompanied by signs of reduced vagal drive and depressed baroreflex gain; while sympathetic vasomotor modulation appears similar in controls and smokers. Data are consistent with the hypothesis that autonomic alterations may contribute to the increased cardiovascular risk present in smokers.

Atherosclerosis is a major complication of type 2 diabetes. The pathogenesis of this complication is poorly understood, but it clearly involves production in the vascular wall of macrophage (Mo) lipoprotein lipase (LPL). Mo LPL is increased in human diabetes. Peripheral factors dysregulated in diabetes, including glucose and free fatty acids (FAs), may contribute to this alteration. We previously reported that high glucose stimulates LPL production in both J774 murine and human Mo. In the present study, we evaluated the direct effect of FAs on murine Mo LPL expression and examined the involvement of peroxisome proliferator-activated receptors (PPARs) in this effect. J774 Mo were cultured for 24 h with 0.2 mmol/l unsaturated FAs (arachidonic [AA], eicosapentaenoic [EPA], and linoleic acids [LA]) and monounsaturated (oleic acid [OA]) and saturated FAs (palmitic acid [PA] and stearic acid [SA]) bound to 2% bovine serum albumin. At the end of this incubation period, Mo LPL mRNA expression, immunoreactive mass, activity, and synthetic rate were measured. Incubation of J774 cells with LA, PA, and SA significantly increased Mo LPL mRNA expression. In contrast, exposure of these cells to AA and EPA dramatically decreased this parameter. All FAs, with the exception of EPA and OA, increased extra- and intracellular LPL immunoreactive mass and activity. Intracellular LPL mass and activity paralleled extracellular LPL mass and activity in all FA-treated cells. In Mo exposed to AA, LA, and PA, an increase in Mo LPL synthetic rate was observed. To evaluate the role of PPARs in the modulatory effect of FAs on Mo LPL gene expression, DNA binding assays were performed. Results of these experiments demonstrate an enhanced binding of nuclear proteins extracted from all FA-treated Mo to the peroxisome proliferator-response element (PPRE) consensus sequence of the LPL promoter. PA-, SA-, and OA-stimulated binding activity was effectively diminished by immunoprecipitation of the nuclear proteins with anti-PPAR-alpha antibodies. In contrast, anti-PPAR-gamma antibodies only significantly decreased AA-induced binding activity. Overall, these results provide the first evidence for a direct regulatory effect of FAs on Mo LPL and suggest a potential role of PPARs in the regulation of Mo LPL gene expression by FAs.

BACKGROUND

Restriction, a highly specific ribotoxin made by the fungus Aspergillus restrictus, cleaves a single phosphodiester bond in the 28S RNA of eukaryotic ribosomes, inhibiting protein synthesis. The sequence around this cleavage site is a binding site for elongation factors, and is conserved in all cytoplasmic ribosomes. The catalytic mechanism of restrictocin and the reasons for its high substrate specificity are unknown. No structure has been determined for any other member of the Aspergillus ribotoxin family.

RESULTS

The crystal structure of restrictocin was determined at 2.1 A resolution by single isomorphous replacement and anomalous scattering techniques, and refined to 1.7 A resolution using synchrotron Laue data. The structural core of the protein, in which a three-turn alpha helix is packed against a five-stranded antiparallel beta sheet, can be well aligned with that of ribonuclease T1. Large positively charged peripheral loops near the active site construct a platform with a concave surface for RNA binding.

CONCLUSIONS

Restriction appears to combine the catalytic components of T1 ribonucleases with the base recognition components of Sa ribonucleases. Modeling studies using an NMR structure of an RNA substrate analog suggest that the tertiary structure of the substrate RNA is important in protein-RNA recognition, fitting closely into the concavity of the presumed binding site. We speculate that the large 39-residue loop L3, which has similarities to loops found in lectin sugar-binding domains, may be responsible for restrictocin's ability to cross cell membranes.

Carbohydrate ligands are important mediators of biomolecular recognition. Microcalorimetry has found the complex-type N-linked glycan core pentasaccharide beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man-(1-->6)]-Man to bind to the lectin, Concanavalin A, with almost the same affinity as the trimannoside, Man-alpha-(1-->6)-[Man-alpha-(1-->3)]-Man. Recent determination of the structure of the pentasaccharide complex found a glycosidic linkage psi torsion angle to be distorted by 50 degrees from the NMR solution value and perturbation of some key mannose-protein interactions observed in the structures of the mono- and trimannoside complexes. To unravel the free energy contributions to binding and to determine the structural basis for this degeneracy, we present the results of a series of nanosecond molecular dynamics simulations, coupled to analysis via the recently developed MM-GB/SA approach (Srinivasan et al., J. Am. Chem. Soc. 1998, 120:9401-9409). These calculations indicate that the strength of key mannose-protein interactions at the monosaccharide site is preserved in both the oligosaccharides. Although distortion of the pentasaccharide is significant, the principal factor in reduced binding is incomplete offset of ligand and protein desolvation due to poorly matched polar interactions. This analysis implies that, although Concanavalin A tolerates the additional 6 arm GlcNAc present in the pentasaccharide, it does not serve as a key recognition determinant.

Staphylococcus aureus is a ubiquitous gram-positive bacterium that can cause superficial to serious systemic infections in animals and humans. Here we report the development of a plant infection model to study the pathogenesis of this bacterium. Three global regulatory mutants, RN6911 (agr-), ALC 488 (sarA-) ALC 842 (sarA-/agr-) and an alpha-toxin mutant defective in biofilm formation (DU1090) which are attenuated in animal pathogenesis, were also attenuated in their ability to infect plants, suggesting that these regulators that mediate synthesis of virulence factors essential for animal pathogenesis are also required for plant pathogenesis. Further, using Arabidopsis plants altered in defense responses such as the transgenic lines NahG [defective in salicylic acid (SA) accumulation], and 35S-LOX2- (defective in jasmonic acid production and hyper-accumulator of SA), and mutants ics1 (depleted in SA accumulation), and npr1-1 (non-expressor of pathogenesis-related protein) we show that resistance of Arabidopsis to typical plant pathogens and the animal pathogen S. aureus is conserved and is mediated by SA. The data presented here suggest that Arabidopsis thaliana resistance to S. aureus is mediated either by a direct effect of SA on the pathogen, specifically one that affects the attachment/aggregate formation on the root surface and reduces the pathogen's virulence, or by SA-dependent, NPR1-independent host responses.

BACKGROUND

Simpson-Angus Scale (SAS) is an established instrument for neuroleptic-induced parkinsonism (NIP), but its statistical properties have been studied insufficiently. Some shortcomings concerning its content have been suggested as well. According to a recent report, the widely used SAS mean score cut-off value 0.3 of for NIP detection may be too low. Our aim was to evaluate SAS against DSM-IV diagnostic criteria for NIP and objective motor assessment (actometry).

METHODS

Ninety-nine chronic institutionalised schizophrenia patients were evaluated during the same interview by standardised actometric recording and SAS. The diagnosis of NIP was based on DSM-IV criteria. Internal consistency measured by Cronbach's alpha, convergence to actometry and the capacity for NIP case detection were assessed.

RESULTS

Cronbach's alpha for the scale was 0.79. SAS discriminated between DSM-IV NIP and non-NIP patients. The actometric findings did not correlate with SAS. ROC-analysis yielded a good case detection power for SAS mean score. The optimal threshold value of SAS mean score was between 0.65 and 0.95, i.e. clearly higher than previously suggested threshold value.

CONCLUSIONS

We conclude that SAS seems a reliable and valid instrument. The previously commonly used cut-off mean score of 0.3 has been too low resulting in low specificity, and we suggest a new cut-off value of 0.65, whereby specificity could be doubled without loosing sensitivity.

BACKGROUND

Scleroderma is an autoimmune disorder of unknown cause. Previous epidemiological studies have suggested some regional clustering and associations with occupations involving exposure to silica dusts and hydrocarbons.

OBJECTIVE

To determine: (i) prevalence and incidence of scleroderma in South Australia (SA), a state with a stable population living in well-defined urban, rural and industrial regions, (ii) hospital separation rates, (iii) cumulative survival rates, (iv) gender and disease subclassification, (v) geographical residency and occupations, (vi) familial associations and (vii) age at onset versus age-specific incidence rate.

METHODS

Creation of the South Australian Scleroderma Register (SASR) to identify demographics and clinical and serological features of all scleroderma patients resident in SA.

RESULTS

Scleroderma occurs in South Australia with a point prevalence of 23 per 10(5) and an incidence of approximately 1/15th of this. However, this prevalence is high compared with other regional world studies. Scleroderma is predominantly a female disease, with most patients having the limited form of scleroderma characterized by the presence of the anticentromere antibody and a mean survival of 27.6 years. In contrast, diffuse scleroderma is a less benign disease occurring at an early age of onset and has a mean survival of 9.6 years. Scleroderma occurs throughout SA without regional localization. Weak associations are seen in males, but not females, with occupations involving possible environmental exposure to silica or hydrocarbons. Scleroderma rarely occurs in families.

CONCLUSIONS

No strong genetic or environmental influences were found to account for the relatively common occurrence of scleroderma in SA. The age at onset versus age-specific incidence curve suggests that scleroderma can be considered as a stochastic illness involving a number of random events occurring in a predisposed population. These random events may involve mutations of pivotal somatic genes.

OBJECTIVE

Social support may benefit mental and physical well-being, but most research has focused on the receipt, rather than the provision, of social support. We explored the potentially beneficial effects of support giving by examining the neural substrates of giving support to a loved one. We focused on a priori regions of interest in the ventral striatum and septal area (SA) because of their role in maternal caregiving behavior in animals.

METHODS

Twenty romantic couples completed a functional magnetic resonance imaging session in which the female partner underwent a scan while her partner stood just outside the scanner and received unpleasant electric shocks.

RESULTS

Support giving (holding a partner's arm while they experienced physical pain), compared with other control conditions, led to significantly more activity in the ventral striatum, a reward-related region also involved in maternal behavior (p values < .05). Similar effects were observed for the SA, a region involved in both maternal behavior and fear attenuation. Greater activity in each of these regions during support giving was associated with greater self-reported support giving effectiveness and social connection (r values = 0.55-0.64, p values < .05). In addition, in line with the SA's role in fear attenuation (presumably to facilitate caregiving during stress), increased SA activity during support giving was associated with reduced left (r = -0.44, p < .05) and right (r = -0.42, p < .05) amygdala activity.

CONCLUSIONS

Results suggest that support giving may be beneficial not only for the receiver but also for the giver. Implications for the possible stress-reducing effects of support giving are discussed.

Heterotrimeric G(i) proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of G(i) proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of Galpha(i2) or Galpha(i1/3) protein in Galpha(i2)-knockout (Galpha(i2)-/-) or Galpha(i1/3)-knockout (Galpha(i1/3)-/-) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (MPhi) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF-alpha and IFN-gamma in splenocytes from Galpha(i2)-/- mice compared with wild-type (WT) mice. Also, LPS-induced TNF-alpha was increased in splenocytes from Galpha(i1/3)-/- mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-alpha, IL-10, and thromboxane B(2) (TxB(2)) production was decreased in MPhi harvested from Galpha(i2)-/- mice. Also, LPS-induced production of IL-10 and TxB(2) was decreased in MPhi from Galpha(i1/3)-/- mice. In subsequent in vivo studies, TNF-alpha levels after LPS challenge were significantly greater in Galpha(i2)-/- mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated Galpha(i2)-/- mice compared with WT mice. These data suggest that G(i) proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli.

This study assessed the ability of executive cognitive functioning (ECF) to predict reactive aggression in boys at high and low risk for substance abuse using a 2-year prospective design. ECF is defined as the self-regulation of goal-directed behavior. Reactive aggression involves impulsive hostile reactions committed with little forethought. ECF was measured using five neuropsychological tests in 198 10- to 12-year-old boys with (SA+) and without (SA-) a paternal history of substance abuse/dependence. Reactive aggression was measured, 2 years later, using a composite index of items derived from two self-report measures. It was hypothesized that ECF would predict reactive aggression, and that this relation would be stronger for the SA+ compared with the SA- boys. SA+ subjects demonstrated lower ECF scores and higher reactive aggression scores, compared with SA- controls. ECF predicted reactive aggression in the SA+ group (beta = 0.37, p = 0.001), but not in the SA- group (beta = 0.09, p = NS). This suggests that compromised ECF may be a risk factor for reactive aggression in SA+ youth. The hypothesis that the relation between ECF and reactive aggression is a manifestation of a mild dysfunction of the prefrontal cortex is discussed.

BACKGROUND

Dynamic instability of coronary atherosclerotic plaque results in the development of both unstable angina and myocardial infarction. The aim of the study was to investigate the dynamics of serum concentrations of tumour necrosis factor (TNF)alpha, interleukin (IL)-10, and IL-2 in patients with myocardial infarction (MI) and unstable angina (UA) as compared to stable angina (SA) patients and healthy volunteers.

METHODS

A total of 189 patients with coronary artery disease (CAD) were studied: 100 patients with SA (class II/III according to CCS), 57 patients with UA (Braunwald class IIIB; determinations at 6, 24, and 48 h after chest pain), and 32 patients with MI (determinations at admission, on the 7th and 30th days after MI). Twenty healthy volunteers acted as controls.

RESULTS

Serum TNFalpha levels were elevated in all CAD groups (SA: 17.3+/-4; UA: 18.7+/-4; MI: 22.0+/-3 pg/ml; p<0.001) in comparison to the controls (8.3+/-1.4 pg/ml). However, the highest values were characteristic of MI patients, especially values obtained at admission (p<0.01 versus SA and UA). Mean serum concentrations of IL-2 were significantly higher in patients with MI and UA (89.6+/-40; 87.0+/-24 pg/ml, respectively; p<0.01) when compared to SA and the control group (58.3+/-49; and 51.5+/-39, respectively). Serum IL-10 levels were also higher in MI and UA patients. Levels of IL-2 and IL-10 measured following chest pain in unstable patients, as well as their consecutive determinations in MI patients did not show any change dynamics, that is, they were persistently elevated.

CONCLUSIONS

When compared to stable CAD and healthy subjects, acute coronary syndromes are associated with long-term increase of serum concentrations of pro- and anti-inflammatory cytokines. It seems likely that sudden CAD progression leading to acute coronary syndromes is triggered/accompanied by prolonged immune activation.