Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding.

A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.     

Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5′-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4–6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.     

Immunophenotyping of mitotic cells from long-term cultures of chorionic villi

Chromosomal mosaicism in chorionic villus samples (CVS) may arise from different sources, such as clonal diversity within the chorionic tissue or contamination with maternal cells. To determine the origin of karyotyped cells, we compared the immunocytochemical features of mitotic cells in CVS long-term cultures with histological sections of their tissue of origin, i.e. chorionic villi. Immunolabelling of intermediate filaments specific for epithelial cells (cytokeratin) and mesenchymal cells (vimentin) established that mitoses yielded from CVS long-term cultures indeed stem from independently growing clones derived from both the epithelial and mesenchymal parts of the chorionic villi. Thus, mosaicism in CVS cultures may reflect true genetic heterogeneity within the biopsy. However, epithelial chorionic cells undergo in vitro metaplasia leading to co-expression of cytokeratins and vimentin. Fetal-specific immune markers (-HCG and SP1-glycoprotein) are not reliably expressed in CVS cell culture.     

Seagrass ecosystem multifunctionality under the rise of a flagship marine megaherbivore

Large grazers (megaherbivores) have a profound impact on ecosystem functioning. However, how ecosystem multifunctionality is affected by changes in megaherbivore populations remains poorly understood. Understanding the total impact on ecosystem multifunctionality requires an integrative ecosystem approach, which is especially challenging to obtain in marine systems. We assessed the effects of experimentally simulated grazing intensity scenarios on ecosystem functions and multifunctionality in a tropical Caribbean seagrass ecosystem. As a model, we selected a key marine megaherbivore, the green turtle, whose ecological role is rapidly unfolding in numerous foraging areas where populations are recovering through conservation after centuries of decline, with an increase in recorded overgrazing episodes. To quantify the effects, we employed a novel integrated index of seagrass ecosystem multifunctionality based upon multiple, well-recognized measures of seagrass ecosystem functions that reflect ecosystem services. Experiments revealed that intermediate turtle grazing resulted in the highest rates of nutrient cycling and carbon storage, while sediment stabilization, decomposition rates, epifauna richness, and fish biomass are highest in the absence of turtle grazing. In contrast, intense grazing resulted in disproportionally large effects on ecosystem functions and a collapse of multifunctionality. These results imply that (i) the return of a megaherbivore can exert strong effects on coastal ecosystem functions and multifunctionality, (ii) conservation efforts that are skewed toward megaherbivores, but ignore their key drivers like predators or habitat, will likely result in overgrazing-induced loss of multifunctionality, and (iii) the multifunctionality index shows great potential as a quantitative tool to assess ecosystem performance. Considerable and rapid alterations in megaherbivore abundance (both through extinction and conservation) cause an imbalance in ecosystem functioning and substantially alter or even compromise ecosystem services that help to negate global change effects. An integrative ecosystem approach in environmental management is urgently required to protect and enhance ecosystem multifunctionality.     

Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor

Secreted forms of the sialoglycoprotein designated cellularprion protein (PrP^C) have been identified that cannot be explainedby alternative splicing. We report that secreted forms of PrP^Cderive from precursors that are bound to the plasma membraneby glycoinositol phospholipid (GPI) anchors. Secreted PrP^C slowlyappeared in the culture medium of metabolically radiolabelledcells after incubations of 8—24 h. Digestion of nascentPrP^C with phosphatidylinositol-specific phospholipase C (PIPLC)prevented the appearance of secreted PrP^C. Secreted PrP^c partitionedinto the aqueous phase of Triton X–114 like PIPLC-releasedPrP^C. While the M_r of PIPLC-released PrP^C was reduced 2–4kDa after treatment with aqueous hydroflouric acid, which removesthe entire GPI anchor modification, the M_r of secreted PrP^Cwas unchanged. Both PIPLC-released and secreted PrP^c were recognizedby antiserum raised against a synthetic C-terminal peptide correspondingto residues 220–233 (amino acid 231 is the site of GPIattachment). We conclude that GPI-anchored PrP^C is post-translationallyprocessed to remove most, if not all, of the GPI modificationand then shed into culture medium. Whether PrP^C is shed afterproteolysis near the C-terminus remains to be established. Aminority of PrP^C in normal Syrian hamster brain partitionedinto the aqueous phase of Triton X–114 like shed PrP^Csuggesting physiological significance. post-translational prion protein secretion sialoglycoprotein     

Refined structure of bovine carbonic anhydrase III at 2.0 Å resolution

The three-dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 Å resolution by both constrained and restrained structure-factor least squares refinement. The current crystallographic R-value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the “deep water” position at the bottom of the cavity and we sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley-Liss, Inc.     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota strain R7 (chemotype Rd1P−)

Abstract Rabbit polyclonal antibodies against the lipopolysaccharide (LPS) of the Rd₁P⁻ mutant strain R7 of Salmonella minnesota were serologically characterized using R7 LPS, dephosphorylated LPS, deacylated LPS, deacylated, dephosphorylated and reduced LPS, and synthetic partial structures. The latter comprised partial structures of the core region of Rd₁P⁻ LPS bound to the β 1 → 6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or artificial glycoconjugates comprised of the synthetic oligosaccharides coupled to bovine serum albumin. Using a passive hemolysis and an enzyme immunoassay, absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core region and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The majority of phosphate-independent antibodies was directed against the trisaccharide l -glycero-α- d -manno-heptopyranose(1 → 3)- l -glycero-α- d -manno-heptopyranose(1 → 5)3-deoxy- d -manno-octulosonic acid. Antibodies against the 1 → 3- and 1 → 7-linked heptose disaccharides and against a single heptose were also detected, however, with low titers. No antibodies were found which required the presence of fatty acids.     

Treatment-resistant depression: definition,prevalence, detection,management, and investigational interventions

Treatment-resistant depression (TRD) is common and associated with multiple serious public health implications. A consensus definition of TRD with demonstrated predictive utility in terms of clinical decision-making and health outcomes does not currently exist. Instead, a plethora of definitions have been proposed, which vary significantly in their conceptual framework. The absence of a consensus definition hampers precise estimates of the prevalence of TRD, and also belies efforts to identify risk factors, prevention opportunities, and effective interventions. In addition, it results in heterogeneity in clinical practice decision-making, adversely affecting quality of care. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have adopted the most used definition of TRD (i.e., inadequate response to a minimum of two antidepressants despite adequacy of the treatment trial and adherence to treatment). It is currently estimated that at least 30% of persons with depression meet this definition. A significant percentage of persons with TRD are actually pseudo-resistant (e.g., due to inadequacy of treatment trials or non-adherence to treatment). Although multiple sociodemographic, clinical, treatment and contextual factors are known to negatively moderate response in persons with depression, very few factors are regarded as predictive of non-response across multiple modalities of treatment. Intravenous ketamine and intranasal esketamine (co-administered with an antidepressant) are established as efficacious in the management of TRD. Some second-generation antipsychotics (e.g., aripiprazole, brexpiprazole, cariprazine, quetiapine XR) are proven effective as adjunctive treatments to antidepressants in partial responders, but only the olanzapine-fluoxetine combination has been studied in FDA-defined TRD. Repetitive transcranial magnetic stimulation (TMS) is established as effective and FDA-approved for individuals with TRD, with accelerated theta-burst TMS also recently showing efficacy. Electroconvulsive therapy is regarded as an effective acute and maintenance intervention in TRD, with preliminary evidence suggesting non-inferiority to acute intravenous ketamine. Evidence for extending antidepressant trial, medication switching and combining antidepressants is mixed. Manual-based psychotherapies are not established as efficacious on their own in TRD, but offer significant symptomatic relief when added to conventional antidepressants. Digital therapeutics are under study and represent a potential future clinical vista in this population.     

Das eigenartige Kopulationsverhalten vonChloromonas saprophila,einer neuen Chlamydomonadacee

Zusammenfassung Chloromonas saprophila n. sp., die in H₂S-haltigem Milieu über verwesendem Laub auftrat, zeichnet sich durch ihr Kopulationsverhalten aus. Die Gameten gleichen jungen vegetativen Zellen und entstehen wie diese zu viert aus einer Mutterzelle. Die Kopulation beginnt bei höherer Individuenzahl unter Gruppenbildung, bei niederer unter Pärchenbildung, Die Geißeln der Gameten sind in den Pärchen zu zweit parallel aneinander gelegt und miteinander verklebt. In den Kopulationsgruppen sind zwei Bündel von Geißeln in entsprechender Zahl vorhanden.Die Gameten verschiedenen Geschlechts stimmen zunächst morphologisch überein, verhalten sich jedoch verschieden: während des Herumschwimmens der Pärchen wird stets der gleiche Gamet vorangetrieben; dieser streift vom Vorderende beginnend seine Membran ab und befestigt sich in der Regel mit seinem Vorderende an der Flanke des behäuteten Gameten; die Geißelpaare trennen sich unterdessen. An der Befestigungsstelle wird die Membran des behäuteten Gameten lokal aufgelöst und sein Protoplast tritt in den des unbehäuteten über.Die reifen Zygoten haben eine glatte, bräunliche Wand und einen kupferroten Inhalt.Der unbehäutete Gamet ist von einer zarten, hyalinen Spezialhülle unbekannter Natur umgeben. Sie zeigt sich auch am Protoplasten des behäuteten dort, wo er sich von der Wand abhebt, und außerdem an den jungen Zygoten und an vegetativen Zellen, bei denen die Membran ausnahmsweise an einzelnen Stellen etwas absteht.