Chiral copper(I)—thiolate clusters in metallothionein and glutathione

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal–thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M₇S₂₀ structure are M₄(S_cys)₁₁ (α domain) and M₃(S_cys)₉ (β domain). Up to 12 Cu(I) ions can displace the 7 Zn²⁺ ions bound per molecule in Zn₇–MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu₁₂S₂₀ structure of Cu₆(S_cys)₁₁ and Cu₆(S_cys)₉ for the α and β domains, respectively. The circular dichroism (CD) spectra recorded as Cu⁺ is added to Zn₇–MT to form Cu₁₂–MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(I); because the Cu(I)–thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)–MT species. Model Cu(I)–thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)–thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)–thiolate clusters in the α and β domains of Cu₁₂–MT are proposed. © 1994 Wiley-Liss, Inc.     

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.     

Size structure of the metazoan community in a Piedmont stream

We characterized the size structure of virtually the entire metazoan community in a fourth order, sandybottomed Piedmont stream during late summer. Our study, the first to sample across all habitat types and sizes of metazoans in an aquatic ecosystem, indicates that at the community level, stream size spectra may be bimodal for the benthos or trimodal when fish are included. Animals spanning 10 orders of magnitude in dry mass (from gastrotrichs to fish) were quantitatively collected from nine habitat types. The bimodal benthic size spectrum was characterized by a meiofaunal component (mostly oligochaetes and micro-crustacea) and a macrobenthic component (mostly the introduced asiatic clam, Corbicula fluminea). Insects contributed little to overall standing crop. Size-specific contribution to whole-community metabolism was assessed using allometric equations for respiration, and we found a distinctly bimodal distribution across the entire metazoan size range, with peaks in the meiofaunal and benthic macrofaunal size ranges. Our bimodal benthic size spectrum is similar to that observed for marine benthos but not to other freshwater benthic systems, possibly because the entire range of habitat types and/or animal sizes were not sampled in the latter. Numerous factors may influence size spectra in stream ecosystems, including local geomorphic (habitat) conditions, water level fluctuations, species introductions, and predation processes.     

Characterization of serological properties of polyclonal antibodies produced against enzymes involved in the L-selective cleavage of hydantoin derivatives

Summary Polyclonal antibodies were produced against the highly purified enzymes L-hydantoinase, hydantoin-racemase and L-N-carbamoylamino acid amidohydrolase of Arthrobacter aurescens DSM 3747. In order to exploit these antibodies for basic research (molecular biology) or bioengineering (process development), the serological properties had to be characterized. Both, the hydantoinase- and carbamoylase-antibodies were observed to be monofunctional, whereas the hydantoin-racemase-antibody was found to be additionally specific against the L-hydantoinase. Monospecificity was realized after affinity chromatography. Investigations on serological crossreactions with several linear- and cyclic amidases (e.g. hydantoinases) as well as hydantoin-racemases are demonstrated in this paper.Deticated to Prof. Dr. Klaus Mosbach on the occation of his 60th birthday.     

The microaerophily and photosensitivity of Propionibacterium acnes

E. MARTIN GRIBBON, J.G. SHOESMITH, W.J. CUNLIFFE AND K.T. HOLLAND. 1994. The effect of oxygen on the in vitro propagation of Propionibacterium acnes was investigated under defined culture conditions. This micro-organism is the predominant bacterial resident within the pilosebaceous follicles of sebum-rich areas of human skin. The organism was grown in continuous culture in defined synthetic medium with glucose as the main carbon-energy source at various air saturation concentrations and in the presence and absence of light. Steady state continuous cultures were achieved at very low oxygen tensions in the presence of light, and at higher levels of oxygen when non-illuminated. Culture biomass yields were higher than those of anaerobic cultures. Bacterial cells were inactivated in the presence of light at high oxygen concentrations because of photosensitization reactions involving excess oxygen and microbial porphyrin species.     

Human bone marrow stromal cells express an osteoblastic phenotype in culture

Summary This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [³H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D₃ by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with⁴⁵Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.     

COVID-19 plasma proteome reveals novel temporal and cell-specific signatures for disease severity and high-precision disease management

Coronavirus disease 2019 (COVID-19) is a systemic inflammatory condition with high mortality that may benefit from personalized medicine and high-precision approaches. COVID-19 patient plasma was analysed with targeted proteomics of 1161 proteins. Patients were monitored from Days 1 to 10 of their intensive care unit (ICU) stay. Age- and gender-matched COVID-19-negative sepsis ICU patients and healthy subjects were examined as controls. Proteomic data were resolved using both cell-specific annotation and deep-analysis for functional enrichment. COVID-19 caused extensive remodelling of the plasma microenvironment associated with a relative immunosuppressive milieu between ICU Days 3–7, and characterized by extensive organ damage. COVID-19 resulted in (1) reduced antigen presentation and B/T-cell function, (2) increased repurposed neutrophils and M1-type macrophages, (3) relatively immature or disrupted endothelia and fibroblasts with a defined secretome, and (4) reactive myeloid lines. Extracellular matrix changes identified in COVID-19 plasma could represent impaired immune cell homing and programmed cell death. The major functional modules disrupted in COVID-19 were exaggerated in patients with fatal outcome. Taken together, these findings provide systems-level insight into the mechanisms of COVID-19 inflammation and identify potential prognostic biomarkers. Therapeutic strategies could be tailored to the immune response of severely ill patients.