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A novel transistor with a graphene base embedded between two n-type silicon emitter and collector layers (graphene-base heterojunction transistor) is fabricated and characterized electrically. The base voltage controlled current of the device flows vertically from the emitter via graphene to the collector. Due to the extremely short transit time for electrons passing the ultimately thin graphene base, the device has a large potential for high-frequency RF applications. The transistor exhibits saturated output currents and a clear modulation of the collector current by means of the graphene base voltage. The vertical transfer current from the emitter via the graphene base to the collector is much lower than expected from device simulations. A comparison of the graphene-base transistor and a reference silicon n-p-n bipolar transistor is performed with respect to the main DC transistor characteristics. A common-emitter gain of larger than one has been achieved for the reference device while the graphene-base transistor so far exhibits a much lower gain. [ABSTRACT FROM AUTHOR]

The effect of sulfide on K+ influx pathways was measured in red blood cells (RBCs) of sulfide-sensitive rainbow trout ( Oncorhynchus mykiss) and sulfide-tolerant crucian carp ( Carassius carassius). In trout RBCs, maximal inhibition of Na+, K+-ATPase was attained at 10 μmol l−1 sulfide and amounted to 32% without being influenced by pH between 6.7 and 8.3. Ouabain-resistant K+ influx in the absence and presence of sulfide was insignificant at pH values between 6.7 and 7.7. At higher pH values ouabain-resistant K+ influx increased, but was inhibited to about 15% by 30 μmol l−1 sulfide. In RBCs of crucian carp neither Na+, K+-ATPase nor ouabain-resistant K+ influx were affected by sulfide concentrations up to 850 μmol l−1. Differences in sulfide-sensitivity of K+ influx between both species can be based upon different properties of the membrane transporter themselves. The reduced Na+, K+-ATPase activity in trout RBCs may also result from a slightly reduced (by 9%) ATP level after sulfide exposure. In addition, intracellular sulfide concentrations were higher in trout RBCs as compared to crucian carp. In trout, intracellular sulfide concentrations reached extracellular levels within 5 min of incubation whereas sulfide concentrations in crucian carp RBCs remained about 2-fold lower than extracellular concentrations. Although the physiological basis of sulfide-insensitive K+ influx in crucian carp RBCs is currently unknown it may contribute to the extremely high sulfide-tolerance of this species. [ABSTRACT FROM AUTHOR]

Background: Acute respiratory distress syndrome (ARDS) in corona virus disease 19 (COVID-19) is triggered by hyperinflammation, thus providing a rationale for immunosuppressive treatments. The Janus kinase inhibitor Ruxolitinib (Ruxo) has shown efficacy in severe and critical COVID-19. In this study, we hypothesized that Ruxo's mode of action in this condition is reflected by changes in the peripheral blood proteome., Methods: This study included 11 COVID-19 patients, who were treated at our center's Intensive Care Unit (ICU). All patients received standard-of-care treatment and n = 8 patients with ARDS received Ruxo in addition. Blood samples were collected before (day 0) and on days 1, 6, and 10 of Ruxo treatment or, respectively, ICU admission. Serum proteomes were analyzed by mass spectrometry (MS) and cytometric bead array., Results: Linear modeling of MS data yielded 27 significantly differentially regulated proteins on day 1, 69 on day 6 and 72 on day 10. Only five factors (IGLV10-54, PSMB1, PGLYRP1, APOA5, WARS1) were regulated both concordantly and significantly over time. Overrepresentation analysis revealed biological processes involving T-cells only on day 1, while a humoral immune response and complement activation were detected at day 6 and day 10. Pathway enrichment analysis identified the NRF2-pathway early under Ruxo treatment and Network map of SARS-CoV-2 signaling and Statin inhibition of cholesterol production at later time points., Conclusion: Our results indicate that the mechanism of action of Ruxo in COVID-19-ARDS can be related to both known effects of this drug as a modulator of T-cells and the SARS-CoV-2-infection., Competing Interests: CS: Consultancy and Research Funding from Hycor Biomedical, Bencard Allergie and Thermo Fisher Scientific; Research Funding from Mead Johnson Nutrition. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Völkel, Tarawneh, Sacher, Bhagwat, Karim, Mack, Wiesmann, Beutel, Hoyer, Keller, Renz, Burchert, Neubauer, Graumann, Skevaki and Mack.)

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

A graphene-based three-terminal barristor device was proposed to overcome the low on/off ratios and insufficient current saturation of conventional graphene field-effect transistors. In this study, we fabricated and analyzed a novel graphene-based transistor, which resembles the structure of the barristor but uses a different operating condition. This new device, termed graphene adjustable-barriers transistor (GABT), utilizes a semiconductor-based gate rather than a metal-insulator gate structure to modulate the device currents. The key feature of the device is the two graphene-semiconductor Schottky barriers with different heights that are controlled simultaneously by the gate voltage. Due to the asymmetry of the barriers, the drain current exceeds the gate current by several orders of magnitude. Thus, the GABT can be considered an amplifier with an alterable current gain. In this work, a silicon-graphene-germanium GABT with an ultra-high current gain ( I D / I G up to 8 × 10 6 ) was fabricated, and the device functionality was demonstrated. Additionally, a capacitance model is applied to predict the theoretical device performance resulting in an on-off ratio above 10 6 , a swing of 87 mV/dec, and a drive current of about 1 × 10 6 A/cm 2 .

Since the first day of life, a newborn has to deal with various pathogens from the environment. While passive immune protection is provided by diaplacental maternal antibodies, the development of cellular immunity is ongoing. A mature immune system should be able not only to defend against pathogens, but should also be able to differentiate between self- and non-self-antigens. Dysregulation in the development of cellular immunity can lead to severe disorders like immunodeficiency, autoimmunity and chronic inflammation. In this review, we explain the role of T cell immunity in antigen detection and summarize the characteristics of a mature TCR repertoire as well as the current state of knowledge about the development of the TCR repertoire in ontogenesis. In addition, methods of assessments are outlined, with a focus on the advantages and disadvantages of advanced methods such as next generation sequencing. Subsequently, we provide an overview of various disorders occuring in early childhood like immunodeficiencies, autoimmunity, allergic diseases and chronic infections and outline known changes in the TCR repertoire. Finally, we summarize the latest findings and discuss current research gaps as well as potential future developments., Competing Interests: For CS: Consultancy and research funding, Hycor Biomedical and Thermo Fisher Scientific; Research Funding, Mead Johnson Nutrition (MJN); Consultancy, Bencard Allergie. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Foth, Völkel, Bauersachs, Zemlin and Skevaki.)

This work describes the fabrication of anisotropically etched, faceted pyramidal structures in amorphous layers of silicon dioxide or glass. Anisotropic and crystal-oriented etching of silicon is well known. Anisotropic etching behavior in completely amorphous layers of silicon dioxide in combination with purely isotropic hydrofluoric acid as etchant is an unexpected phenomenon. The work presents practical exploitations of this new process for self-perfecting pyramidal structures. It can be used for textured silica or glass surfaces. The reason for the observed anisotropy, leading to enhanced lateral etch rates, is the presence of thin metal layers. The lateral etch rate under the metal significantly exceeds the vertical etch rate of the non-metallized area by a factor of about 6-43 for liquid and 59 for vapor-based processes. The ratio between lateral and vertical etch rate, thus the sidewall inclination, can be controlled by etchant concentration and selected metal. The described process allows for direct fabrication of shallow angle pyramids, which for example can enhance the coupling efficiency of light emitting diodes or solar cells, can be exploited for producing dedicated silicon dioxide atomic force microscopy tips with a radius in the 50 nm range, or can potentially be used for surface plasmonics., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Halobacterium salinarum R1 is an extremely halophilic archaeon capable of adhesion and forming biofilms, allowing it to adjust to a range of growth conditions. We have recently shown that living in biofilms facilitates its survival under Cu 2+ and Ni 2+ stress, with specific rearrangements of the biofilm architecture observed following exposition. In this study, quantitative analyses were performed by SWATH mass spectrometry to determine the respective proteomes of planktonic and biofilm cells after exposition to Cu 2+ and Ni 2+ .Quantitative data for 1180 proteins were obtained, corresponding to 46% of the predicted proteome. In planktonic cells, 234 of 1180 proteins showed significant abundance changes after metal ion treatment, of which 47% occurred in Cu 2+ and Ni 2+ treated samples. In biofilms, significant changes were detected for 52 proteins. Only three proteins changed under both conditions, suggesting metal-specific stress responses in biofilms. Deletion strains were generated to assess the potential role of selected target genes. Strongest effects were observed for ΔOE5245F and ΔOE2816F strains which exhibited increased and decreased biofilm mass after Ni 2+ exposure, respectively. Moreover, EPS obviously plays a crucial role in H. salinarum metal ion resistance. Further efforts are required to elucidate the molecular basis and interplay of additional resistance mechanisms., (Copyright © 2020 Völkel, Hein, Benker, Pfeifer, Lenz and Losensky.)

Background: Patients with cystic fibrosis (CF) may suffer from iron deficiency which is a known risk factor for the restless legs syndrome (RLS), however, its prevalence has not yet been investigated in these subjects., Patients and Methods: Adult out-patients with CF (n = 39) and healthy volunteers (n = 32) were recruited for this study. A diagnosis of RLS was made based on the diagnostic criteria established by the International Restless Legs Syndrome Study Group (IRLSSG). The IRLSSG rating scale was used to assess the severity of the disease. Furthermore, in the CF group, parameters of iron metabolism were measured in peripheral venous blood samples., Results: The RLS occurred more frequently in the CF patients than the controls (n = 13/33,3% vs. n = 2/6,3%; p < 0,05). In the CF patients suffering from RLS, the mean score of the IRLSSG rating scale was 17,2 ± 9,4 indicating moderate disease severity. Iron deficiency was present in the majority of the CF patients investigated (n = 33/84,6%), however, serum iron, ferritin and transferrin levels as well as transferrin saturation were similar in those with vs. without RLS., Conclusions: The frequency of the RLS is increased in adult patients with CF. On an average, its severity is moderate and it is not related to iron deficiency as evaluated by serum parameters of iron metabolism., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Halobacterium salinarum R1 is an extremely halophilic archaeon, able to attach to the surface and to form characteristic biofilm structures under physiological conditions. However, the effect of environmental stress factors like heavy metals on biofilms was still unknown. Here, we report on the first insights into H. salinarum biofilm formation when exposed to copper, nickel and zinc and describe the effects of metal ions on the architecture of mature biofilms. We also studied the effects on gene expression in planktonic cells. Investigation of planktonic growth and cell adhesion in the presence of sub-lethal metal concentrations yielded an up to 60% reduced adhesion in case of copper and a significantly enhanced adhesion in case of zinc, whereas nickel treatment had no effect on adhesion. A PMA-qPCR assay was developed to quantify live/dead cells in planktonic cultures and mature biofilms, enabling the investigation of cell vitality after metal exposure. An increased resistance was observed in biofilms with up to 80% in case of copper- and up to 50% in case of zinc exposure compared to planktonic cells. However, nickel-treated biofilms showed no significant increase of cell survival. Microscopic investigation of the architecture of mature biofilms exposed to lethal metal concentrations demonstrated an increased detachment and the formation of large microcolonies after copper treatment, whereas the number of adherent cells increased strongly in nickel-exposed biofilms. In contrast, zinc exposed-biofilms showed no differences compared to the control. Analysis of the expression of genes encoding putative metal transporters by qRT-PCR revealed specific changes upon treatment of the cells with heavy metals. Our results demonstrate diverse effects of heavy metal ions on H. salinarum and imply a metal-specific protective response of cells in biofilms.

Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2-promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding-independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes., (© 2018 Völkel et al.)

Porous calcium phosphate (CaP) materials as bone graft substitutes can be prepared from Ca carbonate biomineral structures by hydrothermal conversion into pseudomorphic CaP scaffolds. The present study aims at furnishing such phosphatized Ca carbonate biomineral (PCCB) materials with antibacterial Ag ions in order to avoid perisurgical wound infections. Prior to this study, PCCB materials with Mg and/or Sr ions incorporated for stimulating bone formation were prepared from coral skeletons and sea urchin spines as starting materials. The porous PCCB materials were treated with aqueous solutions of Ag nitrate with concentrations of 10 or 100 mmol/L, resulting in the formation of Ag phosphate nanoparticles on the sample surfaces through a replacement reaction. The materials were characterized using scanning electron microscopy (SEM) energy-dispersive X-ray spectroscopy (EDS) and X-ray diffractometry (XRD). In contact with Ringer`s solution, the Ag phosphate nanoparticles dissolved and released Ag ions with concentrations up to 0.51 mg/L, as shown by atomic absorption spectroscopy (AAS) analyses. In tests against Pseudomonas aeruginosa and Staphylococcus aureus on agar plates, antibacterial properties were similar for both types of Ag-modified PCCB materials. Concerning the antibacterial performance, the treatment with AgNO₃ solutions with 10 mmol/L was almost as effective as with 100 mmol/L.

Experiments on a thin layer of cohesive wet granular matter under vertical vibrations reveal kink-separated domains that collide with the container at different phases. Due to the strong cohesion arising from the formation of liquid bridges between adjacent particles, the domains move collectively upon vibrations. Depending on the periodicity of this collective motion, the kink fronts may propagate, couple with each other, and form rotating spiral patterns in the case of period tripling or stay as standing wave patterns in the case of period doubling. Moreover, both patterns may coexist with granular "gas bubbles"-phase separation into a liquidlike and a gaslike state. Stability diagrams for the instabilities measured with various granular layer mass m and container height H are presented. The onsets for both types of patterns and their dependency on m and H can be quantitatively captured with a model considering the granular layer as a single particle colliding completely inelastically with the container.

Transcription factors are grouped into families based on sequence similarity within functional domains, particularly DNA-binding domains. The Specificity proteins Sp1, Sp2 and Sp3 are paradigmatic of closely related transcription factors. They share amino-terminal glutamine-rich regions and a conserved carboxy-terminal zinc finger domain that can bind to GC rich motifs in vitro. All three Sp proteins are ubiquitously expressed; yet they carry out unique functions in vivo raising the question of how specificity is achieved. Crucially, it is unknown whether they bind to distinct genomic sites and, if so, how binding site selection is accomplished. In this study, we have examined the genomic binding patterns of Sp1, Sp2 and Sp3 in mouse embryonic fibroblasts by ChIP-seq. Sp1 and Sp3 essentially occupy the same promoters and localize to GC boxes. The genomic binding pattern of Sp2 is different; Sp2 primarily localizes at CCAAT motifs. Consistently, re-expression of Sp2 and Sp3 mutants in corresponding knockout MEFs revealed strikingly different modes of genomic binding site selection. Most significantly, while the zinc fingers dictate genomic binding of Sp3, they are completely dispensable for binding of Sp2. Instead, the glutamine-rich amino-terminal region is sufficient for recruitment of Sp2 to its target promoters in vivo. We have identified the trimeric histone-fold CCAAT box binding transcription factor Nf-y as the major partner for Sp2-chromatin interaction. Nf-y is critical for recruitment of Sp2 to co-occupied regulatory elements. Equally, Sp2 potentiates binding of Nf-y to shared sites indicating the existence of an extensive Sp2-Nf-y interaction network. Our results unveil strikingly different recruitment mechanisms of Sp1/Sp2/Sp3 transcription factor members uncovering an unexpected layer of complexity in their binding to chromatin in vivo.

Objective: The goal of this study was to describe coping strategies and their associations with psychological distress in young adult survivors of childhood cancer., Methods: One hundred and sixty-four childhood cancer survivors, at least 7 years after diagnosis, completed questionnaires assessing demographics, health information, psychological distress, and different ways of coping (return rate: 61%). The Brief Symptom Inventory-18 (BSI-18) and the Post-traumatic Diagnostic Scale's (PDS) eight-item short form were used to measure psychological distress. Coping was assessed with the Cognitive Control Strategies Scale (CCSS), the Illness Perception Questionnaire-Revised (IPQ-R), and the White Bear Suppression Inventory (WBSI)., Results: Higher levels of distress were associated with the female sex, not being in a relationship, and with the presence of medical late effects. These predictors explained 12% of the variance in psychological distress. When coping variables were also entered into the equation, the amount of explained variance increased to 50%. The most important determinants of psychological distress in our sample were a tendency to suppress negative thoughts and a low level of optimism., Conclusion: These results contribute to a better understanding of the correlates of difficulties in long-term psychological adjustment after childhood cancer. Cognitive strategies, which are associated with or may increase the risk for concurrent psychological distress, in specific, avoidance of negative thoughts and a lack of positive future expectations, should be addressed in psychological counseling with survivors suffering from symptoms of distress., (Copyright © 2012 John Wiley & Sons, Ltd.)

Aims: Gastro-oesophageal reflux disease (GERD) is associated with impaired epithelial barrier function. This study was aimed at investigating the role of desmosomal proteins in relation to GERD., Methods and Results: Ninety-five patients with GERD-related symptoms (erosive, n = 51; non-erosive, n = 44) and 27 patients lacking those symptoms were included. Endoscopic and histological characterization of oesophagitis was performed according to the Los Angeles and Ismeil-Beigi criteria, respectively. Multiple biopsies were taken from the oesophageal mucosa of each patient. Gene expression analysis of plakoglobin, desmoglein-1, desmoglein-2 and desmoglein-3 was performed by quantitative real time (RT)-polymerase chain reaction and immunohistochemistry in the oesophageal mucosa. Routine histology revealed specific GERD-related alterations, such as dilatation of intercellular spaces (DIS), basal cell hyperplasia (BCH), and elongation of the papillae, in the oesophageal mucosa of patients with GERD, as compared with controls (all parameters: P < 0.05). All four genes and corresponding proteins were found to be up-regulated by between 1.7 and 8.1-fold (transcript level, P < 0.05; protein level, P < 0.05). Induced gene expression levels of plakoglobin, desmoglein-1 and desmoglein-2 correlated significantly with DIS and BCH., Conclusions: Taken together, the uniform up-regulation of desmosomal genes/proteins in the oesophageal mucosa of patients with GERD supports the concept of architectural and molecular changes in the desmosomal compartment in the pathogenesis of GERD., (© 2011 Blackwell Publishing Ltd.)

Objectives: Secretory leukocyte protease inhibitor (SLPI) serves as a 'defense shield' against serine proteases in inflammation. Gastroesophageal reflux disease (GERD) is associated with chronic inflammation and histomorphological alterations of the gastroesophageal junction and esophageal mucosa. Here, it was investigated whether the presence of GERD was associated with changes of mucosal SLPI expression., Methods: Ninety-five patients with GERD-related symptoms and 27 patients lacking those symptoms were included. Endoscopic and histological evaluation was done according to the Los Angeles and updated Sydney classifications. Multiple biopsies were taken from gastric and esophageal mucosa of each patient for histology, immunohistochemistry (IHC), and molecular analyses. SLPI expression was analyzed by quantitative reverse transcriptase-PCR, enzyme-linked immunoassay, and IHC, and the data were statistically analyzed with respect to endoscopic and clinical parameters., Results: Forty-four patients had nonerosive and 51 erosive reflux diseases, respectively. Histology revealed higher chronic inflammation (P=0.04) and significant alterations of the intercellular spaces, basal cell hyperplasia, and length of papilla (P<0.05) in patients with GERD. Mucosal SLPI levels were comparable among antrum, cardia, and esophagus ranging from 95 to 165 pg/mug protein and were not affected by the presence of GERD, whereas esophageal SLPI-transcript levels were three-fold induced in patients with GERD (P=0.002). IHC identified epithelial cells as major cellular source of mucosal SLPI expression in normal cardiac and esophageal mucosa, whereas infiltrating immune cells contributed to the SLPI expression in chronically inflamed tissue., Conclusion: GERD, a chemically induced inflammation, does not affect mucosal SLPI expression in gastroesophageal mucosa.

The O2 dependence of ouabain-independent K+ transport mechanisms has been studied by unidirectional Rb+ flux analysis in crucian carp red blood cells (RBCs). The following observations suggest that O2 activates K+-Cl- cotransport (KCC) and deactivates Na+-K+-2Cl- cotransport (NKCC) in these cells via separate O2 sensors that differ in their O2 affinity. When O2 tension (PO2) at physiological pH 7.9 was increased from 0 to 1, 4, 21 or 100 kPa, K+ (Rb+) influx was increasingly inhibited, and at 100 kPa amounted to about 30% of the value at 0 kPa. This influx was almost completely Cl- dependent at high and low PO2, as shown by substituting Cl- with nitrate or methanesulphonate. K+ (Rb+) efflux showed a similar PO2 dependence as K+ (Rb+) influx, but was about 4-5 times higher over the whole PO2 range. The combined net free energy of transmembrane ion gradients favoured net efflux of ions for both KCC and NKCC mechanisms. The KCC inhibitor dihydroindenyloxyalkanoic acid (DIOA, 0.1 mM) abolished Cl- -dependent K+ (Rb+) influx at a PO2 of 100 kPa, but was only partially effective at low PO2 (0-1 kPa). At PO2 values between 0 and 4 kPa, K+ (Rb+) influx was further unaffected by variations in pH between 8.4 and 6.9, whereas the flux at 21 and 100 kPa was strongly reduced by pH values below 8.4. At pH 8.4, where K+ (Rb+) influx was maximal at high and low PO2, titration of K+ (Rb+) influx with the NKCC inhibitor bumetanide (1, 10 and 100 microM) revealed a highly bumetanide-sensitive K+ (Rb+) flux pathway at low PO2, and a relative bumetanide-insensitive pathway at high PO2. The bumetanide-sensitive K+ (Rb+) influx pathway was activated by decreasing PO2, with a PO2 for half-maximal activation (P50) not significantly different from the P50 for haemoglobin O2 binding. The bumetanide-insensitive K+ (Rb+) influx pathway was activated by increasing PO2 with a P50 significantly higher than for haemoglobin O2 binding. These results are relevant for the pathologically altered O2 sensitivity of RBC ion transport in certain human haemoglobinopathies.

The effects of pH and O(2) tension on the isotonic ouabain-resistant K(+) (Rb+) flux pathway and on haemoglobin O2 binding were studied in trout red blood cells (RBCs) in order to test for a direct effect of haemoglobin O(2) saturation on K(+) transport across the RBC membrane. At pH values corresponding to in vivo control arterial plasma pH and higher, elevation of the O(2) partial pressure (PO(2)) from 7.8 to 157 mmHg increased unidirectional K(+) influx across the RBC membrane several-fold. At lower extracellular pH values, stimulation of K(+) influx by O(2) was depressed, exhibiting an apparent pK(a) (pK'(a)) for the process of 8.0. Under similar conditions the pK'(a) for acid-induced deoxygenation of haemoglobin (Hb) was 7.3. When trout RBCs were exposed to PO(2) values between 0 and 747 mmHg, O(2) equilibrium curves typical of Hb O(2) saturation were also obtained for K(+) influx and efflux. However, at pH 7.9, the PO(2) for half-maximal K(+) efflux and K(+) influx (P50) was about 8- to 12-fold higher than the P(50) for Hb-O(2) binding. While K(+) influx and efflux stimulation by O(2) was essentially non-cooperative, Hb-O(2) equilibrium curves were distinctly sigmoidal (Hill parameters close to 1 and 3, respectively). O(2)-stimulated K(+) influx and efflux were strongly pH dependent. When the definition of the Bohr factor for respiratory pigments (Phi = delta logP50 x delta pH(-1)) was extended to the effect of pH on O(2)-dependent K(+) influx and efflux, extracellular Bohr factors (Phi(o) of -2.00 and -2.06 were obtained, values much higher than that for Hb (Phi(o) = -0.49). The results of this study are consistent with an O(2) sensing mechanism differing markedly in affinity and cooperativity of O(2) binding, as well as in pH sensitivity, from bulk Hb.

A method for the quantitative determination of sulphaemoglobin (SHb) in a mixture of haemoglobin derivatives by spectral deconvolution is described. SHb formation was studied in haemolysates and in red blood cells of the sulphide-sensitive rainbow trout (Oncorhynchus mykiss) and of the sulphide-tolerant common carp (Cyprinus carpio). Addition of sulphide caused the formation of SHb in haemolysates of both animals. However, haemoglobin from common carp was much less sensitive to sulphide than was trout haemoglobin. The maximal obtainable SHb fraction was approximately 30 % in trout and 10 % in carp haemolysates. In both animals, the SHb fraction increased with increasing Hb and sulphide concentrations up to 100 micromol l(-)(1) and 1 mmol l(-)(1), respectively, and was favoured by a low pH. An increase of temperature between 5 and 25 degrees C strongly increased SHb formation in trout haemolysate. In contrast, temperature changes had almost no effect on SHb production in carp. Within trout red blood cells, approximately 7 % of total haemoglobin was converted to SHb during 60 min of incubation (with 2.5 mmol l(-)(1) sulphide), inducing a 20 % loss of haemoglobin oxygen-saturation. In carp red blood cells incubated under identical conditions, SHb formation was minimal and haemoglobin oxygen-saturation was not affected.

Many aquatic animal species can survive sulfide exposure to some extent through oxidation of the sulfide, which results mainly in thiosulfate. In several species, sulfide oxidation is localized in the mitochondria and is accompanied by ATP synthesis. In addition, blood-based and intracellular compounds can augment sulfide oxidation. The formation of thiosulfate requires oxygen, which results in an increase in oxygen consumption of some species. If not all sulfide is detoxified, cytochrome C oxidase is inhibited. Under these conditions, a sulfide-dependent anaerobic energy metabolism commences.

Sulfide is oxidized in the mitochondria of the lugworm Arenicola marina. Mitochondrial sulfide oxidation is coupled with oxygen consumption and with an equimolar production of thiosulfate [Völkel, S. & Grieshaber, M. K. (1994) Mar. Biol. 118, 137-147]. Mitochondrial respiration in the presence of malate (or succinate) and ADP but without sulfide could be completely inhibited by rotenone, antimycin, cyanide, and sulfide. Only 40% inhibition was achieved by salicylhydroxamic acid. Sulfide oxidation (with sulfide as the only substrate) was fully inhibited by antimycin and by salicylhydroxamic acid but not by rotenone or sulfide. Moreover, sulfide oxidation was 3-4-fold less sensitive to cyanide as compared to normal respiration. The data indicate that sulfide oxidation in A. marina is linked to the respiratory electron transport chain. We suggest that electrons from sulfide enter the respiratory chain via ubiquinone or at the ubiquinol-cytochrome-c oxidoreductase. At sulfide concentrations higher than 10 microM, the cytochrome-c oxidase is blocked and electrons from sulfide are transferred to oxygen via an alternative terminal oxidase.

The immunological responses to subcutaneous bone and cartilage grafts were investigated in rats using phenol oxidase, a leukocyte labelling enzyme. The resulting development of granulomas reflects a cell-mediated immunological process and is proof of the immunogenicity of bone and cartilage. This method is a new criterium for the assessment of the biocompatibility of graft and implant materials.

With the aid of the histochemical phenoloxidase (PO) assay, 12 substances were tested. On the one side they can act as substrates or as inhibitors of phenoloxidase and on the other side as immunogenic or immunosuppressive compounds. In particular the following substances were investigated: o-cresol, Levamisol, prednisolonebisuccinate, Bleocin, siliciumdioxide, 4-methylbrenzcatechol, +-catechine, thiomersal, D-penicillamine, hydrochinonemonomethylether, hydrochinonemonoethylether, and hydrochinonemonobenzylether. No changes in the phenoloxidase activity and in the morphological behaviour of the cells could be detected using Levamisol, prednisolonebisuccinate, siliciumdioxide, and Bleomycin, indicating an immunogenic stimulation or immunosuppressive effect. 4-methylbrenzcatechol is probably transformed into a soluble reaction product by phenoloxidase. It could not clearly demonstrated by the histochemical method, if this substance is used as a true substrate. +-catechine, thiomersal, and D-penicillamine cause strong to complete enzyme inhibition of the phenoloxidase containing cells (POC), which are located in the spleen and gut of the white rat. Hydrochinonemonoethylether, hydrochinonemethylether, and hydrochinonemonobenzylether cause an increased pigment fermentation, but they are not used as substrates for the PO.