Stability of α-chymotrypsin conjugated with poly (ethylene glycols) and proxanols at high temperature and in watercosolvent mixtures

Summary -Chymotrypsin has been modified with poly(ethylene glycols) and proxanols, block-copolymers of poly(propylene oxide) and poly(ethylene oxide). These conjugates were several-fold more thermostable and showed high catalytic activity at elevated concentrations of water-miscible organic cosolvents (alcohols and dimethyl sulfoxide) which caused inactivation of free (non-modified) -chymotrypsin.     

Gliotoxin penetrates and impairs the integrity of the human blood-brain barrier in vitro

Cerebral fungal infections represent an important public health concern, where a key element of pathophysiology is the ability of the fungi to cross the blood-brain barrier (BBB). Yet the mechanism used by micro-organisms to cross such a barrier and invade the brain parenchyma remains unclear. This study investigated the effects of gliotoxin (GTX), a mycotoxin secreted by Aspergillus fumigatus, on the BBB using brain microvascular endothelial cells (BMECs) derived from induced pluripotent stem cells (iPSCs). We observed that both acute (2 h) and prolonged (24 h) exposure to GTX at the level of 1 μM or higher compromised BMECs monolayer integrity. Notably, acute exposure was sufficient to disrupt the barrier function in iPSC-derived BMECs, resulting in decreased transendothelial electrical resistance (TEER) and increased fluorescein permeability. Further, our data suggest that such disruption occurred without affecting tight junction complexes, via alteration of cell-matrix interactions, alterations in F-actin distribution, through a protein kinase C-independent signaling. In addition to its effect on the barrier function, we have observed a low permeability of GTX across the BBB. This fact can be partially explained by possible interactions of GTX with membrane proteins. Taken together, this study suggests that GTX may contribute in cerebral invasion processes of Aspergillus fumigatus by altering the blood-brain barrier integrity without disrupting tight junction complexes.     

The trimeric coiled‐coil HSBP1 protein promotes WASH complex assembly at centrosomes

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5β1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled‐coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.     

Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe

Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-(bromoacetyl)-2-(dimethylamino)naphthalene (BADAN), 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and alpha-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of the BADAN-modified enzyme is characterized by an S50 of 18.2 +/- 0.7, compared with the value of 2.2 +/- 0.3 for the ANF-induced spin transition, thus revealing an additional low-affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer.     

Presence of oxygen-consuming ribonucleotide reductase in corrinoid-deficientPropionibacterium freudenreichii

Corrinoid-deficient Propionibacterium freuden- reichii subsp. shermanii showed adenosylcobalamin-(AdoCbl)-independent ribonucleotide reductase (RNR) activity in the presence of air. Increasing the incubation time with free access of O₂ led to an increase in RNR activity. As polarographic estimations of O₂ uptake demonstrated, AdoCbl-independent RNR activity (with ADP as substrate) in a cell-free system of corrinoid-deficient P. freudenreichii was accompanied by specific molecular oxygen consumption. The activity was not inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCCP). The activity was present in the cytoplasmic membrane-free soluble fraction of the cell extract, and it was inhibited by hydroxyurea. Manganese ions were important for the cell division of corrinoid-deficient P. freudenreichii and stimulated RNR activity after 8-hydroxyquinoline or EDTA treatment of the cell extract. We therefore concluded that P. freudenreichii is able to form DNA (deoxyribosylic precursors) using AdoCbl-dependent ribonucleotide reductase and also with an alternative AdoCbl-independent molecular-oxygen-consuming RNR system. Received: 29 May 1995 / Accepted: 14 August 1995     

Changes in the biota of Chany Lake along a salinity gradient

Relationships among salinity and diversity, abundance, biomass of major biological components of Chany Lake (western Siberia, Russia) are examined across a salinity gradient. As salinity increased from 0.8 to 6.4 g l⁻¹, the species richness of aquatic vascular plants decreased from 16 to 2 species, of phytoplankton from 98 to 52 species, and of zooplankton from 61 to 16 species, but changes in species diversity of zoobenthos were negligible. Guest Editor: John M. Melack Saline Waters and their Biota     

Effect of Low-Intensity Pulsed Electric Fields on the Respiratory Activity and Electrosurface Properties of Bacteria

A change in the respiratory activity (RA) of the resistant to cyanide bacteria Pseudomonas fluorescens under the pulsed electric field treatment has been found experimentally. The bacteria reaction to the field treatment (rectangular impulses with the duration of 1 ms, voltage of 20 V, and frequency of 100 Hz) varied from the RA suppression of active culture to restoration and even stimulation of RA in cells with the decreased respiratory function, caused by the cyanide introduction. The interconnection of energetic membrane processes with the electrosurface properties of biocolloids has been demonstrated: The electrokinetic potential had been varied simultaneously with the respiratory activity change. Theoretical analysis of the experimental data indicates the change of quantity of active respiratory centers in cells under the influence of the pulsed electric field. These changes have reversible character. Depending on the state of bacteria, the processes of inhibition or restoration of the respiratory centers by the pulsed electric field can be observed.