The heparin binding domain of S-protein/vitronectin binds to complement components C7, C8, and C9 and perforin from cytolytic T-cells and inhibits their lytic activities

S-Protein/vitronectin is a serum glycoprotein that inhibits the lytic activity of the membrane attack complex of complement, i.e., of the complex including the proteins C5b, C6, C7, C8, and C9n. We show that intact S-protein/vitronectin or its cyanogen bromide generated fragments also inhibit the hemolysis mediated by perforin from cytotoxic T-cells at 45 and 11 microM, respectively. The glycosaminoglycan binding site of S-protein/vitronectin is responsible for the inhibition, since a synthetic peptide corresponding to a part of this highly basic domain (amino acid residues 348-360) inhibits complement- as well as perforin-mediated cytolysis. In the case of C9, the synthetic peptide binds to the acidic residues occurring in its N-terminal cysteine-rich domain (residues 101-111). Antibodies raised against this particular segment react 25-fold better with the polymerized form of C9 as compared with its monomeric form, indicating that this site becomes exposed only upon the hydrophilic-amphiphilic transition of C9. Since the cysteine-rich domain of C9 has been shown to be highly conserved in C6, C7, and C8 as well as in perforin, the inhibition of the lytic activities of these molecules by S-protein/vitronectin or by peptides corresponding to its heparin binding site may be explained by a similar mechanism.     

Effect of feeding boxes on the behavior of stereotyping amur tigers (Panthera tigris altaica) in the Zurich Zoo,Zurich, Switzerland

The stereotyped pacing shown by the two Amur tigers in the Zurich Zoo was hypothesized as being caused by permanently frustrated appetitive foraging behavior. Several electrically controlled feeding boxes were installed and access to each box was possible only twice a day for 15 min at semi‐random times. The boxes had to be opened actively by the tigers. Two trials were carried out: one with solitary confinement, and one with paired confinement. During box feeding, the female's stereotyped pacing was significantly reduced from 16% (solitary confinement, conventional feeding) and 7% (paired confinement, conventional feeding) to 1% (solitary confinement) and less than 0.01% (paired confinement) of the daily observed time. The female's sleeping increased significantly in both solitary and paired confinement. The male only showed a significant reduction in stereotyped pacing behavior when kept with the female (conventional feeding: 10%; box feeding: <0.01% of the daily observed time). On days with a box‐feeding regime in paired confinement, the male spent 25% (83 min) of the observed time with active behavior at the feeding boxes. The results support the hypothesis that permanently frustrated appetitive foraging behavior causes stereotyped pacing in adult tigers. Zoo Biol 21:573–584, 2002. © 2002 Wiley‐Liss, Inc.     

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Kinetics of nitrite oxidation in two Nitrobacter species grown in nitrite-limited chemostats

The influence of growth rate, the presence of acetate and variation in the dissolved oxygen concentration on the kinetics of nitrite oxidation was studied in suspensions of intact cells of Nitrobacter winogradskyi and Nitrobacter hamburgensis. The cells were grown in nitrite-limited chemostats at different dilution rates under chemolithotrophic and mixotrophic conditions. Growth of N. hamburgensis in continuous culture was dependent on the presence of acetate. Acetate hardly affected the maximal nitrite oxidation rate per cell (V _max), but displayed a distinctly negative effect on the saturation constants for nitrite oxidation (K _m ) of both Nitrobacter species. This effect was reversible; when acetate was removed from the suspensions the K _m -values for nitrite oxidation returned to their original values. A reduction of the dissolved oxygen concentration from 100% to 18% air saturation slightly decreased the V _max of chemolithotrophically grown N. winogradskyi cells, whereas a 2.3 fold increase was observed with mixotrophically grown cells of N. hamburgensis. It is suggested that the large variation in K _m encountered in field samples could be due to this observed phenotypic variability. The V _max per cell is not a constant, but apparently is dependent on growth rate and environmental conditions. This implies that potential nitrite oxidation activity and numbers of cells are not necessarily related. Considering their kinetic characteristics, it is unlikely that N. hamburgensis is able to compete succesfully with N. winogradskyi for limiting amounts of nitrite under mixotrophic conditions. However, at reduced partial oxygen tensions, N. hamburgensis may become the better competitor.     

Physicochemical characterization of human S-protein and its function in the blood coagulation system.

S-protein, the main inhibitor of the assembly of the membrane attack complex of complement, was isolated from human plasma by a simple purification procedure, which includes barium citrate adsorption, ammonium sulphate precipitation, chromatography on DEAE-Sephacel and Blue Sepharose and gel filtration on Sephacryl S-200. The homogeneous protein (sedimentation coefficient 4.6 S) was obtained in approx. 5% yield relative to its concentration in plasma, which was found to be 0.3-0.5 mg/ml. The final product did not cross-react with antisera against complement proteins or other proteinase inhibitors of human plasma. On polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, S-protein migrated as a single-chain band with an apparent Mr of 74000 under non-reducing conditions and as a doublet of Mr 78000 and 65000 upon reduction. In plasma or serum S-protein also existed in two forms of corresponding Mr values, as was evidenced by an immunoblot enzyme-linked immunosorbent assay technique. S-protein was found to be an acidic glycoprotein with 10% (W/W) carbohydrate content and several isoelectric points in the range pH 4.75-5.25, and it contained one free thiol group per molecule of protein. The functional properties of S-protein in the complement system were demonstrated by its ability to inhibit complement-dependent cell lysis in a concentration-dependent manner (Ki 0.6 microM) and by its incorporation into the nascent SC5b-7 complex. A new function for S-protein could be revealed in the blood coagulation system. The slow progressive inhibition of thrombin by antithrombin III was not affected by S-protein, whereas the purified protein interfered with the fast inactivation of thrombin clotting as well as amidolytic activity by antithrombin III-heparin complex. The acceleration of this inhibition reaction by heparin was counteracted by S-protein, indicating the ability of S-protein to neutralize heparin activity.     

IL-1 alpha or tumor necrosis factor-alpha stimulate release of three NAP-1/IL-8-related neutrophil chemotactic proteins in human dermal fibroblasts

Human dermal fibroblasts in culture secrete three protein-like neutrophil chemotactic factors, when stimulated either with human rIL-1 alpha or IL-1 beta; not, however, after incubation with LPS. These three fibroblast-derived neutrophil-activating proteins (FINAP) could be purified by subsequently performed reversed phase and size exclusion HPLC. By high resolution SDS-PAGE, all the proteins were shown to migrate with an Mr of 6,700 (alpha-FINAP), 3,600 (beta-FINAP), and 5,300 (gamma-FINAP). All purified cytokine preparations were found to be chemotactic for human neutrophils. In addition, all FINAP induced release of lysosomal enzymes in neutrophils. Deactivation of chemotaxin-elicitable enzyme release showed cross-desensitization of all FINAP with NAP-1/IL-8. Western blot analysis of alpha-FINAP by using mAb against neutrophil-activating protein (NAP)-1/IL-8 reveals immunologic cross-reactivity with NAP-1/IL-8. By amino-terminal amino acid sequence analysis alpha-FINAP could be identified as the 77-residue extended form of NAP-1/IL-8 containing the 79-residue form as a minor contaminant. Whereas beta-FINAP has been found to be a truncation product of alpha-FINAP, gamma-FINAP shows identity with authentic melanoma growth stimulatory activity with respect to retention time upon reversed phase HPLC, high resolution SDS-PAGE, and biologic properties, as well as amino-terminal amino acid sequence. These data show that human dermal fibroblasts may actively participate in inflammatory reactions by secretion of proinflammatory cytokines.     

The Urokinase Receptor Is a Major Vitronectin-Binding Protein on Endothelial Cells

We have previously demonstrated that vitronectin (VN), a morphoregulatory protein in the vessel wall, is internalized and translocated to the subendothelial matrix by an integrin-independent mechanism (J. Histochem. Cytochem.41, 1823–1832, 1993). The cell surface component which mediates the initial contact of VN with endothelial cells is defined here. The specific binding of VN to endothelial cells demonstrated the following properties: a threefold increase after phorbol ester treatment; 85% inhibition by pretreatment of cells with phosphatidylinositol–phospholipase C to release glycolipid-anchored surface proteins; a 90% inhibition by urokinase (u-PA) receptor blocking antibody. u-PA increased VN binding to cells due to an eightfold increase in the affinity of VN for the u-PA receptor. Structure–function studies showed that the amino-terminal fragment of u-PA, devoid of any proteolytic activity, mediated this effect. Active plasminogen activator inhibitor-1 (PAI-1), but not inactivated PAI-1, inhibited VN binding to cells and displaced VN that was prebound to endothelial cell monolayers. Similarly, VN binding to purified (immobilized) u-PA receptor, but not to integrin, was enhanced by u-PA and inhibited by PAI-1. Hence, the binding of soluble VN to endothelial cell surfaces is mediated by the u-PA receptor, and the relative concentrations of u-PA and PAI-1 are able to regulate the strength of this interaction. Endothelial cell adhesion to immobilized VN was found to be integrin-mediated without any involvement of the VN–uPA-receptor system. Hence, the interaction of VN with the u-PA receptor may be involved in the regulation of cellular processes necessary for endothelial cell invasion and migration at VN-rich extracellular matrix sites.     

Real-time monitoring of biomass during Escherichia coli high-cell-density cultivations by in-line photon density wave spectroscopy

An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µ_s′ with CDW was observed in the range of 5–69 g L⁻¹ (R² = 0.98). The growth rates calculated based on µ_s′ were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µ_s′ as a function of the absorption coefficient µ_a allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

Prevalence and trends of common mental disorders from 2007-2009 to 2019-2022: results from the Netherlands Mental Health Survey and Incidence Studies (NEMESIS), including comparison of prevalence rates before vs. during the COVID-19 pandemic

Up-to-date information on the prevalence and trends of common mental disorders is relevant to health care policy and planning, owing to the high burden associated with these disorders. In the first wave of the third Netherlands Mental Health Survey and Incidence Study (NEMESIS-3), a nationally representative sample was interviewed face-to-face from November 2019 to March 2022 (6,194 subjects; 1,576 interviewed before and 4,618 during the COVID-19 pandemic; age range: 18-75 years). A slightly modified version of the Composite International Diagnostic Interview 3.0 was used to assess DSM-IV and DSM-5 diagnoses. Trends in 12-month prevalence rates of DSM-IV mental disorders were examined by comparing these rates between NEMESIS-3 and NEMESIS-2 (6,646 subjects; age range: 18-64 years; interviewed from November 2007 to July 2009). Lifetime DSM-5 prevalence estimates in NEMESIS-3 were 28.6% for anxiety disorders, 27.6% for mood disorders, 16.7% for substance use disorders, and 3.6% for attention-deficit/hyperactivity disorder. Over the last 12 months, prevalence rates were 15.2%, 9.8%, 7.1%, and 3.2%, respectively. No differences in 12-month prevalence rates before vs. during the COVID-19 pandemic were found (26.7% pre-pandemic vs. 25.7% during the pandemic), even after controlling for differences in socio-demographic characteristics of the respondents interviewed in these two periods. This was the case for all four disorder categories. From 2007-2009 to 2019-2022, the 12-month prevalence rate of any DSM-IV disorder significantly increased from 17.4% to 26.1%. A stronger increase in prevalence was found for students, younger adults (18-34 years) and city dwellers. These data suggest that the prevalence of mental disorders has increased in the past decade, but this is not explained by the COVID-19 pandemic. The already high mental disorder risk of young adults has particularly further increased in recent years.