Quantitative proteomic analysis of formalin-fixed, paraffin-embedded clear cell renal cell carcinoma tissue using stable isotopic dimethylation of primary amines.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most abundant resource of archived human specimens in pathology. Such tissue specimens are emerging as a highly valuable resource for translational proteomic studies. In quantitative proteomic analysis, reductive di-methylation of primary amines using stable isotopic formaldehyde variants is increasingly used due to its robustness and cost-effectiveness. RESULTS: In the present study we show for the first time that isotopic amine dimethylation can be used in a straightforward manner for the quantitative proteomic analysis of FFPE specimens without interference from formalin employed in the FFPE process. Isotopic amine dimethylation of FFPE specimens showed equal labeling efficiency as for cryopreserved specimens. For both FFPE and cryopreserved specimens, differential labeling of identical samples yielded highly similar ratio distributions within the expected range for dimethyl labeling. In an initial application, we profiled proteome changes in clear cell renal cell carcinoma (ccRCC) FFPE tissue specimens compared to adjacent non-malignant renal tissue. Our findings highlight increased levels of glyocolytic enzymes, annexins as well as ribosomal and proteasomal proteins. CONCLUSION: Our study establishes isotopic amine dimethylation as a versatile tool for quantitative proteomic analysis of FFPE specimens and underlines proteome alterations in ccRCC.

[Cross stability in conventional shoes by the use of spring steel insoles: a pedobarographic effect study with observational application]. / Querstabilität im Konfektionsschuh durch Federstahleinlagen: Eine pedobarographische Wirkungsstudie mit Anwendungsbeobachtung.

BACKGROUND: From an orthopedic point of view, wearing conventional ready to wear shoes negatively affects the front transverse arch of the human foot by forcing it into an unnatural inverted position. OBJECTIVES: The aim of the present orthopedic application study was to conduct a standardized assessment of the biomechanical effect of a newly developed, longitudinally flexible and cross stable spring steel insole by means of pedobarographic measurements and by means of a supplementary questioning of the participants. MATERIAL AND METHODS: In order to do this a total of 33 healthy adult participants were recruited in summer 2013. The randomized, blinded and controlled main study compared the cross stable insole made from spring steel with a flat, ordinary control insole made from ethylene vinyl acetate by means of pedobarographic in-shoe measurements at the forefoot. Additionally, the subjectively perceived effect of the steel insole with respect to comfort, restricted mobility and pain was assessed in a randomized, blinded and controlled ancillary study, using a cross-over design. RESULTS: Both the plantar peak pressure and the plantar force-time integral were significantly higher with the spring steel insole, especially in the central forefoot. In the subsequent test phase lasting several weeks during which the participants were asked to wear the spring steel insole, they rarely complained about problems and in particular did not report negative effects regarding comfort, restricted mobility and pain compared to the ordinary control insoles. CONCLUSION: The present study was conducted according to high methodological standards and proved for the first time that the tested spring steel insoles have a positive effect on the human forefoot. The cross stability increases the pressure in the median ball area, prevents the unnatural inverted position of the forefoot and thus creating an effect which is comparable to walking barefoot. As the participants did not judge this orthopedic effect of the cross stability as being uncomfortable, such an insole could be used a millionfold as a primary prevention in conventional shoes.

Endocrine disruptors and Leydig cell function.

During the past decades, a large body of information concerning the effects of endocrine disrupting compounds (EDCs) on animals and humans has been accumulated. EDCs are of synthetic or natural origin and certain groups are known to disrupt the action of androgens and to impair the development of the male reproductive tract and external genitalia. The present overview describes the effects of the different classes of EDCs, such as pesticides, phthalates, dioxins, and phytoestrogens, including newly synthesized resveratrol analogs on steroidogenesis in Leydig cells. The potential impact of these compounds on androgen production by Leydig cells during fetal development and in the adult age is discussed. In addition, the possible role of EDCs in connection with the increasing frequency of abnormalities in reproductive development in animals and humans is discussed.

Functional properties of failing human ventricular myocytes.

Reduced peak systolic Ca2+ and slow decay of the Ca2+ transient are common features of the end-stage failing human ventricular myocyte and are thought to underlie abnormal ventricular contractility in congestive heart failure (CHF). Individual changes in the expression or activity of Ca2+ transport proteins of the sarcoplasmic reticulum (SR Ca2+ ATPase, SERCa) or the sarcolemmal (sodium-calcium exchanger, NCX) have not always been observed in CHF and cannot per se consistently explain these Ca2+ transient defects. We review recent data that suggests that the normal balance of transport activities of SERCa and NCX is deranged in failing human myocytes. We hypothesize that an increase in the NCX/SERCa transport capacity in failing myocytes can explain the abnormal Ca2+ homeostasis of the failing human ventricular myocyte.

Design of oligolactone-based scaffolds for bone tissue engineering.

Novel difunctional oligolactone macromers have been synthesized by ring-opening oligomerization of various lactones (L-lactide, glycolide, p-dioxanone) in the presence of suitable diols (propane-1,2-diol, dianhydro-D-glucitol) and subsequent endcapping of the formed oligolactones with methacrylate moieties. Based on these macromers, two fabrication procedures were developed to fabricate highly porous scaffolds and the material properties including in vitro biodegradation behaviour of the resulting polymeric scaffolds were investigated. Preliminary in vitro studies on the cytocompatibility of the fabricated scaffolds and on osteoblast cultivation on the optimized polymeric materials demonstrated that the oligolactide based polymer networks possess an excellent biocompatibility and that they are promising candidates as scaffolds in bone tissue engineering.

Increased basal contractility of cardiomyocytes overexpressing protein kinase C epsilon and blunted positive inotropic response to endothelin-1.

OBJECTIVE: Protein kinase C (PKC) is thought to be involved in the regulation of the mammalian cardiac excitation-contraction coupling process by vasoactive peptides like endothelin-1 (ET-1). However, the demonstration of a causal link between activation of specific PKC isoforms and the increase in contractility mediated by ET-1 is still inferential. METHODS: By means of adenovirus-mediated gene transfer, we specifically overexpressed PKC epsilon in cultured adult rabbit ventricular myocytes (Ad-PKC epsilon). Myocyte shortening and [Ca2+]i transients under basal and ET-1-stimulated conditions were measured in Ad-PKC epsilon and Ad-LacZ control transfected cells. RESULTS: Infection with Ad-PKC epsilon resulted in a strong, virus dose-dependent increase in PKC epsilon protein levels, whereas protein expression of other PKC isoforms remained unchanged. Using a multiplicity of infection of 100 plaque-forming units/myocyte, basal and cofactor-dependent PKC epsilon kinase activity was increased 28- and 90-fold, respectively, when compared to control. Myocyte basal fractional shortening and [Ca2+]i transient amplitude were both increased by 21% (P < 0.05 each) in Ad-PKC epsilon transfected myocytes when compared to Ad-LacZ transfected control myocytes. The positive inotropic effect of ET-1 in control myocytes was markedly blunted in PKC epsilon-overexpressing myocytes. CONCLUSION: Specific overexpression of PKC epsilon in rabbit ventricular myocytes increases basal myocyte contractility and [Ca2+]i transients, and modifies their responsiveness to ET-1.

The maxi-K channel opener BMS-204352 attenuates regional cerebral edema and neurologic motor impairment after experimental brain injury.

Large-conductance, calcium-activated potassium (maxi-K) channels regulate neurotransmitter release and neuronal excitability, and openers of these channels have been shown to be neuroprotective in models of cerebral ischemia. The authors evaluated the effects of postinjury systemic administration of the maxi-K channel opener, BMS-204352, on behavioral and histologic outcome after lateral fluid percussion (FP) traumatic brain injury (TBI) in the rat. Anesthetized Sprague-Dawley rats (n = 142) were subjected to moderate FP brain injury (n = 88) or surgery without injury (n = 54) and were randomized to receive a bolus of 0.1 mg/kg BMS-204352 (n = 26, injured; n = 18, sham), 0.03 mg/kg BMS-204352 (n = 25, injured; n = 18, sham), or 2% dimethyl sulfoxide (DMSO) in polyethylene glycol (vehicle, n = 27, injured; n = 18, sham) at 10 minutes postinjury. One group of rats was tested for memory retention (Morris water maze) at 42 hours postinjury, then killed for evaluation of regional cerebral edema. A second group of injured/sham rats was assessed for neurologic motor function from 48 hours to 2 weeks postinjury and cortical lesion area. Administration of 0.1 mg/kg BMS-204352 improved neurologic motor function at 1 and 2 weeks postinjury (P < 0.05) and reduced the extent of cerebral edema in the ipsilateral hippocampus, thalamus, and adjacent cortex (P < 0.05). Administration of 0.03 mg/kg BMS-204352 significantly reduced cerebral edema in the ipsilateral thalamus (P < 0.05). No effects on cognitive function or cortical tissue loss were observed with either dose. These results suggest that the novel maxi-K channel opener BMS-204352 may be selectively beneficial in the treatment of experimental TBI.

Improving CD4+ lymphocyte counts in HIV-infected hemophilia patients. A favorable prognostic indicator?

CD4+ lymphocyte counts of 91 HIV+ hemophilia patients were monitored for a mean of 4 years (range: 15-69 months). CD4+ lymphocytes decreased in 55 but increased in 36 patients over time. The CD4+ cell increases were persistent in 5 patients, whereas they fluctuated in 31. Of the 36 patients with increasing CD4+ counts 3 developed AIDS and 1 LAS. The other 32 patients were clinically asymptomatic (CDC II), but had immunological abnormalities, such as increased serum neopterin (N = 18) and impaired in vitro T cell responses to pooled allogenic stimulator cells (N = 15) or mitogens (N = 18). In contrast, of the 55 patients whose CD4+ cells decreased, 24 developed AIDS and 5 ARC (P less than 0.0005). Only 2 of these 55 patients had normal mitogen stimulation in vitro and normal serum neopterin levels.

The novel compound LOE 908 attenuates acute neuromotor dysfunction but not cognitive impairment or cortical tissue loss following traumatic brain injury in rats.

Experimental traumatic brain injury (TBI) initiates massive disturbances in Ca2+ concentrations in the brain that may contribute to neuronal damage. Intracellular Ca2+ may be elevated via influx through voltage-operated cation channels, ligand-gated ionotropic channels, and store-operated cation channels (SOCs). In the present study, we evaluated the neurobehavioral and histological effects of acute posttraumatic administration of (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di[2-(2 ,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908), a broad spectrum inhibitor of voltage-operated cation channels and SOCs. Male Sprague-Dawley rats (n = 53) were trained in the Morris water maze, anesthetized (60 mg/kg pentobarbital, i.p.), and subjected to lateral fluid percussion brain injury (2.5-2.7 atm; n = 38) or surgery without injury (n = 15). At 15 min postinjury, animals were randomized to receive intravenous administration of either a high dose of LOE 908 (4 mg/kg bolus followed by 160 mg/kg over 24 h; n = 13), a low dose of LOE 908 (2 mg/kg bolus followed by 80 mg/kg over 24 h; n = 12), or vehicle (n = 13). Uninjured controls received the high dose of LOE 908 (n = 8) or vehicle (n = 7). Treatment with either dose of LOE 908 significantly improved neuromotor function at 48 h postinjury when compared to vehicle treatment. Although a significant deficit in visuospatial memory was observed in brain-injured animals at this timepoint when compared to uninjured animals, neither dose of LOE 908 attenuated injury-induced cognitive dysfunction. Histological evaluation revealed that neither dose of LOE 908 affected cortical lesion size at 48 h postinjury. These data suggest that broad spectrum cation channel blockers may be beneficial in the treatment of neurological motor dysfunction when administered in the acute posttraumatic period.

The erythrocyte-perfused "working heart" model: hemodynamic and metabolic performance in comparison to crystalloid perfused hearts.

A brief period of ischemia was used to evaluate an erythrocyte-enriched Krebs-Henseleit (KH) buffer (n=8) compared to KH only (n=8) in an isolated working rabbit heart. Experimental protocol was as follows: preischemic baseline, 5 min of global ischemia followed by 45 min of reperfusion. Preischemic heart rate was identical, coronary flow was significantly lower (2.7 versus 5.6 mL/min/g wet wt, p<0.01), the other hemodynamic and biochemical values were significantly higher in erythrocyte-perfused hearts: aortic flow 23.5 versus 12.0, p<0.01; cardiac output 26.2 versus 17.6, p<0.01; all in mL/min/g wet wt; dp/dt max 1286 versus 997 mmHg/s, p<0.01; myocardial oxygen consumption 3.5 versus 2.3 micromol/min/g wet wt, p<0.05. During early reperfusion, in the erythrocyte-perfused hearts, coronary flow further increased (p<0.003), the other hemodynamic parameters returned to baseline values in both groups. High-energy phosphates showed significantly higher values (ATP 2.0+/-0.1 versus 1.3+/-0.1, p<0.05; CrP 2.0+/-0.2 versus 1.6+/-0.1, p<0.05 all in micromol/g wct wt), water content was significantly lower (81% versus 74%, p<0.05) in erythrocyte-perfused hearts. It can be concluded that the erythrocyte-perfused working heart model provides excellent oxygenation, leading to superior hemodynamic and metabolic performance. Additionally, in the erythrocyte-perfused hearts preservation of coronary flow reserve underlines the physiological competency of this preparation.

Negative images of bacilli and mycobacterial infection: a study of fine-needle aspiration smears from lymph nodes in patients with AIDS.

In patients with the acquired immunodeficiency syndrome (AIDS), four cases of mycobacterial infection in which bacilli appeared as cylindrical, nonstaining "negative images" have been previously described. These may have been extracellular or within histiocyte cytoplasm, and they have been described in aspirations from liver, lymph node, and bone marrow and in bronchoalveolar lavage fluid. We report three additional examples of this finding in fine-needle aspirations from lymph nodes in AIDS patients infected with Mycobacterium avium-intracellulare. Our findings support the concept that these negative images of bacilli are diagnostic of mycobacterial infection. Air-dried Romanovsky-stained material is required for their identification.

Origin, development and regulation of human Leydig cells.

Sex steroids are crucial regulators of sexual differentiation and the proper development of secondary sex characteristics and patterns of sexual behavior. Since Leydig cells are the primary major producers of these steroid hormones, maintenance of the normal functions of these cells determines the reproductive capacity and fertility of males. The present minireview discusses recent findings concerning endocrine and paracrine regulation of the proliferation, differentiation and involution of human Leydig cells. The physiology and function of the two distinct fetal and adult populations of human Leydig cells are described, with particular focus on the paracrine environment that triggers their differentiation and functional maturation. The roles of established and more recently discovered paracrine regulators of this maturation, including insulin-like factor 3, platelet-derived growth factor-alpha, desert hedgehog, ghrelin and leptin are considered. A brief description of the origin, ontogenesis and functional markers of human fetal and adult Leydig cells is presented.

Development of a bioresorbable self-hardening bone adhesive based on a composite consisting of polylactide methacrylates and beta-tricalcium phosphate.

In this work, a novel bioresorbable bone adhesive based on radically polymerizable polylactide with methacrylate endgroups known from polymethylmethacrylate (PMMA) cements and varying amounts of bioresorbable/biodegradable lactide moieties was developed. The swelling and degradation properties as well as the hardening time, viscosity, and adhesion properties (tension and shear resistance) were subsequently measured in vitro and optimized. For a broad use in surgery the handling properties, the shelf life and the storage temperature are important issues. The finally developed material consists of three substances that have to be mixed to start the reaction: a highly viscous mixture of oligomers and two beta-tricalcium phosphate (beta-TCP, Cerasorb) powders with the radical starter and the promoter. The material has a processing time of 2 min and is completely cured after another minute. The tension and shear resistance of the material is 3.1-13.9 MPa that will decrease by storing the substance in a humid atmosphere. Degradation experiments showed a mass loss of 20-35% during the first 5 weeks. Tests with MC3T3-E1 cells showed an increase of the alkaline phosphatase activity over a period of 14 days. The mechanical and handling properties and the in vitro data are showing a promising biomaterial for bone regeneration.

[Transmission of the human immunodeficiency virus by a dry heat-treated Factor VIII concentrate?]. / Ubertragung von Human-Immunodeficiency-Virus (HIV) durch trocken-hitzebehandelte Faktor-VIII-Gerinnungspräparate?

Short presentation of the common procedures to avoid transmission of human-immunodeficiency-virus (HIV) by hemoderivates especially clotting-factor-preparations. The stepwise seroconversion (ELISA, IFT, Western-blot) of HIV is shown in a 7 5/12 ys old boy with hemophilia A after administration of a dry-heated factor VIII-preparation. Seven similar observations were reported in the literature. On the other hand HIV-seroconversion could not be observed during treatment with wet-heated factor VIII-preparations. In consequence only wet-heated factor VIII-preparations and factor IX-preparations respectively should be administered to hemophiliacs without HIV-antibodies. By this precaution transmission of non-A, non-B-hepatitis may be avoided simultaneously.

Elimination of plasmids by enoxacin and ofloxacin at near inhibitory concentrations.

The abilities of the 4-quinolones enoxacin and ofloxacin, inhibitors of DNA gyrase subunit A, to eliminate plasmids from Escherichia coli have been studied in a narrow concentration range just below the MIC. These compounds cured most efficiently at the highest concentration which still allows cell growth and produced 20% to 100% plasmid-free cells, depending upon the plasmid tested. Higher concentrations were required to eliminate plasmids from a gyrANalr strain, consistent with their higher MICs, but maximal curing frequencies were similar to those obtained with the Nals strain. Kinetics of plasmid elimination indicated that plasmid loss occurred by inhibition of plasmid replication, which seems to be somewhat more sensitive to the action of 4-quinolones than chromosome replication. Low or high curing frequencies with a given curing agent seem to be a property of the plasmid tested.

Inhibition of RNA- and DNA-synthesis by citrinin and its effects on DNA precursor-metabolism in V79-E cells.

1. The RNA synthesis of V79-E cells was inhibited by the mycotoxin citrinin time- and concentration-dependently. 2. Among the different RNA species mainly the rRNA synthesis was found to be inhibited by 200 microM citrinin. 3. At different precursor concentrations DNA synthesis was inhibited by citrinin after 30 min at least whereas labelling of the acid soluble fractions was found to be 3-fold higher than in untreated cells. 4. Remarkable perturbation of the DNA precursor metabolism, including release of precursor into the medium, was found to occur during citrinin treatment.

Inhibition of R-plasmid transfer in Escherichia coli by 4-quinolones.

Inhibition of transfer of four conjugative R plasmids by ciprofloxacin, enoxacin, norfloxacin, ofloxacin, and pipemidic acid was investigated in an Escherichia coli mating system. The absolute concentrations needed for inhibition of conjugation varied from 0.12 microgram/ml for ciprofloxacin to 16 micrograms/ml for pipemidic acid, but the relationship to the MICs for the parent strains was identical for all substrates. Concentrations for a 90% reduction of transconjugants were in the range of one to six times the MIC for the parent strains, which also had lethal effects on donors and recipients. A similar effect on conjugation was found with chloramphenicol. These observations question the specificity of transfer inhibition by quinolones and cast doubt on the clinical importance of such an effect.