Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes.

Enzymatic catalysis and homogeneous catalysis offer complementary means to address synthetic challenges, both in chemistry and in biology. Despite its attractiveness, the implementation of concurrent cascade reactions that combine an organometallic catalyst with an enzyme has proven challenging because of the mutual inactivation of both catalysts. To address this, we show that incorporation of a d(6)-piano stool complex within a host protein affords an artificial transfer hydrogenase (ATHase) that is fully compatible with and complementary to natural enzymes, thus enabling efficient concurrent tandem catalysis. To illustrate the generality of the approach, the ATHase was combined with various NADH-, FAD- and haem-dependent enzymes, resulting in orthogonal redox cascades. Up to three enzymes were integrated in the cascade and combined with the ATHase with a view to achieving (i) a double stereoselective amine deracemization, (ii) a horseradish peroxidase-coupled readout of the transfer hydrogenase activity towards its genetic optimization, (iii) the formation of L-pipecolic acid from L-lysine and (iv) regeneration of NADH to promote a monooxygenase-catalysed oxyfunctionalization reaction.

The mode of interaction between Vitis and Plasmopara viticola Berk. & Curt. Ex de Bary depends on the host species.

In order to obtain insight into host responses to grapevine downy mildew (Plasmopara viticola), we compared pathogen development on a panel of Vitis species from North America, Asia and Europe. Leaf discs from different host species were inoculated in parallel, and the colonisation of the mesophyll was visualised by aniline blue staining and quantified with respect to infection incidence and mycelial growth. In parallel, the morphology of guard cells was screened for the presence of an internal cuticular rim after staining with acridine orange and using low-temperature scanning electron microscopy. We observed three response patterns: (i) inhibition of pathogen development early after attachment of zoospores; (ii) successful colonisation of the mesophyll by the pathogen; and (iii) aberrant development, where the pathogen does not attach to guard cells, but produces hyphae on the leaf surface without formation of viable sporangiophores. Inhibition is observed in the North American and Siberian species, successful colonisation prevails in the European hosts, and surface hyphae are found on non-Siberian Asiatic species. We propose that the interaction between host and pathogen is under control of specific signals that have been subject to evolutionary diversification.

Membrane translocation and oligomerization of hBok are triggered in response to apoptotic stimuli and Bnip3.

hBok is a human pro-apoptotic member of the Bcl-2 family. By fluorescence in situ hybridization and in silico analysis, hBok was found to be located on chromosome 2q37.3. Its expression was detected in various organs and several hormonally regulated cancer cells. Expression of hBok was shown to be upregulated in estrogen-dependent breast cancer by estrogen deprivation and in myocardial cells during hypoxia. Confocal laser scanning microscopy examinations and subcellular fractionation studies showed that hBok was distributed in both the cytosol and intracellular membranes of healthy cells. Upon overexpression of hBok or stimulation of apoptosis, hBok became integrated into the membrane. Furthermore, apoptosis and oligomerization were promoted by BH3-only proteins, such as Bid, Bnip3 and p53, but prevented by BFL-1. hBok was found to interact with Bnip3. Our findings suggest that functional BH3-only proteins facilite the oligomerization and insertion of hBok into the membrane to activate it.

Ultrastructural distribution of the S100A1 Ca2+-binding protein in the human heart.

Impaired calcium homeostasis and altered expression of Ca2+-binding proteins are associated with cardiomyopathies, myocardial hypertrophy, infarction or ischemia. S100A1 protein with its modulatory effect on different target proteins has been proposed as one of potential candidates which could participate in these pathological processes. The exact localization of S100A1 in human heart cells on the ultrastructural level accompanied with biochemical determination of its target proteins may help clarify the role of S100A1 in heart muscle. In the present study the distribution of the S100A1 protein using postembedding (Lowicryl K4M) immunocytochemical method in human heart muscle has been determined quantitatively, relating number of antigen sites to the unit area of a respective structural component. S100A1 antigen sites have been detected in elements of sarcoplasmic reticulum (SR), in myofibrils at all levels of sarcomere and in mitochondria, the density of immunolabeling at Z-lines being about 3 times and at SR more than 5 times higher than immunolabeling of remaining structural components. The presence of the S100A1 in SR and myofibrils may be related to the known target proteins for S100A1 at these sites.

The LIM homeobox gene ceh-14 confers thermosensory function to the AFD neurons in Caenorhabditis elegans.

In Caenorhabditis elegans three pairs of neurons, AFD, AIY, and AIZ, play a key role in thermosensation. The LIM homeobox gene ceh-14 is expressed in the AFD thermosensory neurons. ceh-14 mutant animals display athermotactic behaviors, although the neurons are still present and differentiated. Two other LIM homeobox genes, ttx-3 and lin-11, function in the two interneurons AIY and AIZ, respectively. Thus, the three key thermosensory neurons are specified by three different LIM homeobox genes. ceh-14 ttx-3 lin-11 triple mutant animals have a basic cryophilic thermotaxis behavior indicative of a second thermotaxis pathway. Misexpression of ceh-14 in chemosensory neurons can restore thermotactic behavior without impairing the chemosensory function. Thus, ceh-14 confers thermosensory function to neurons.

Characterization of an immortalized human granulosa cell line (COV434).

We have investigated the biological characteristics of an immortalized granulosa cell line (COV434), which may be used to study follicular and oocyte maturation in vitro. Granulosa cell function was defined as consisting of three distinct properties: (i) production of 17beta-oestradiol in response to follicle stimulating hormone (FSH); (ii) presence of specific molecular markers of apoptosis enabling the induction of follicular atresia; and (iii) capacity to form intercellular connections with cells surrounding an oocyte. The addition of FSH to the culture medium supplemented with 10% fetal calf serum and 4-androstene-3,17-dione resulted in proliferation of the COV434 granulosa cells and in an increased synthesis of 17beta-oestradiol, indicating the presence of the FSH receptor and cytochrome P450 aromatase in these cells. The receptor for luteinizing hormone (LH) was undetectable. Similar expression of various apoptosis-associated genes was found in COV434 granulosa cells and in granulosa cells of patients stimulated with gonadotrophins for in-vitro fertilization, thus indicating that the immortalized COV434 granulosa cells were able to sustain apoptosis. Multiple intercellular connections were formed during co-culture of COV434 granulosa cells with cumulus cells containing an immature oocyte but not with cumulus cells devoid of an oocyte. Detailed morphological analysis of the intercellular connections with scanning electron microscopy and confocal light microscopy demonstrated the presence of long slender structures. It is concluded that the immortalized human granulosa cell line COV434 may be useful for experimental studies on follicular development.

Nuclear export of recombinant baculovirus nucleocapsids through small pore or nuclear-pore-like structure in Sf9 cells.

Translocation of baculovirus nucleocapsids (45 nm in diameter, approximate length of 280-300 nm) from nucleoplasm to cytoplasm was studied morphologically using cryofixation and gold labeled wheat germ agglutinin (WGA-gold) during recombinant Autographa californica nuclear polyhedrosis virus infection in Sf9 cells. Nucleocapsids formed in the nucleoplasm migrated into protrusions of the nuclear envelope, but not into nuclear pore complexes. We found cross-like membranous structures. Small pores seemed to be in the protruding nuclear double membranes. The middle piece of a nucleocapsid was located within the small pore whereas the upper part was in the cytoplasm. Other nucleocapsids were situated within pores without colocalization of WGA-gold in the nuclear envelope. These results suggest that baculovirus nucleocapsids use small pores in the nuclear-derived membranes or incomplete nuclear pores in the nuclear envelope to migrate from the nucleoplasm to the cytoplasm, but not complete nuclear pore complexes proper.

Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy.

The Escherichia coli hns gene, which encodes the nucleoid protein H-NS, was deprived of its natural promoter and placed under the control of the inducible lambda PL promoter. An hns mutant yielding a protein (H-NS delta 12) with a deletion of four amino acids (Gly112-Arg-Thr-Pro115) was also obtained. Overproduction of wild-type (wt) H-NS, but not of H-NS delta 12, resulted in a drastic loss of cell viability. The molecular events and the morphological alterations eventually leading to cell death were investigated. A strong and nearly immediate inhibition of both RNA and protein synthesis were among the main effects of overproduction of wt H-NS, while synthesis of DNA and cell wall material was inhibited to a lesser extent and at a later time. Upon cryofixation of the cells, part of the overproduced protein was found in inclusion bodies, while the rest was localized by immunoelectron microscopy to the nucleoids. The nucleoids appeared condensed in cells expressing both forms of H-NS, but the morphological alterations were particularly dramatic in those overproducing wt H-NS; their nucleoids appeared very dense, compact and almost perfectly spherical. These results provide direct evidence for involvement of H-NS in control of the organization and compaction of the bacterial nucleoid in vivo and suggest that it may function, either directly or indirectly, as transcriptional repressor and translational inhibitor.

Conjugational junctions: morphology of specific contacts in conjugating Escherichia coli bacteria.

F-plasmid-mediated bacterial conjugation was studied with hfr (traDts) and tra I mutant Escherichia coli donor strains. This allowed us to observe a statistically significant number of conjugation-specific contacts by video and electron microscopy. Single mating events between E. coli were observed in real time by video-enhanced light microscopy. Conjugation in vivo takes place by initial contact formation via pili, followed by direct and transient wall-to-wall contact, during which DNA is transferred and disaggregated. Electron microscopic observations of the contact zone between donor and recipient bacteria were made by thin sectioning of mating pairs that were arranged in monolayers. We defined the conjugation-specific contact found in stabilized mating pairs as the conjugational junction. Within this junction no specific substructure such as plasma bridges by fusion could be detected during transfer of DNA.

Escherichia coli DNA-binding protein H-NS is localized in the nucleoid.

Electron microscope localization of the 15.4-kDa DNA-binding protein H-NS was carried out in Escherichia coli cells subjected to cryosubstitution followed by immuno-labelling with the protein A/gold technique. Three types of E. coli cells were used: (1) "normal" cells growing exponentially at 37 degrees C; (2) "cold-shocked" cells two hours after the shift from 37 degrees C to 10 degrees C; and (3) cells in which an expression vector had been induced to overproduce H-NS. The results clearly indicate that in all 3 cases, the vast majority of the molecules reacting with anti-H-NS antibodies are localized within the nucleoid and at the border between the nucleoid and the ribosome-rich cytoplasm, which supports the premise that H-NS is implicated in the condensation of the nucleoid.

Removal of background label in immunocytochemistry with the apolar Lowicryls by using washed protein A-gold-precoupled antibodies in a one-step procedure.

Antiserum mixed with protein A-gold produces aggregates that can be washed by centrifugation and redissociated by ultrasonication. These precoupled antibodies are used for immunocytochemical labeling in a one-step procedure and reveal a very low background on sections of material embedded in polar and apolar Lowicryls. Controls have shown that the background label stems from serum components with only one protein A binding site and is discarded during the wash steps of the precoupling procedure.

Intracellular location of the histonelike protein HU in Escherichia coli.

Immunocytochemical labeling of thin sections of cryosubstituted, Lowicryl-embedded Escherichia coli cells with protein A-colloidal gold was used to study the structural organization of the bacterial nucleoid. We found that the histonelike protein HU was not associated with the bulk DNA in the nucleoid but was located in areas of the cell where metabolically active DNA is associated with ribosomes and where single-stranded DNA, RNA polymerase, and DNA topoisomerase I were also located. The resolution of the methods used did not allow us to decide whether HU was associated either with ribosomes or with transcriptionally active DNA, nor could we demonstrate interaction of HU with either.

The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions.

The surface of thin sections of aldehyde-fixed biological material shows a specimen-related relief of 2-6 nm with Lowicryl. Epon sections are about three times smoother. The relief is the consequence of thin-sectioning being in reality a cleavage. Epitopes are supposed to be laid open (or set free) because cleavage follows the interfaces between protein and Lowicryl. We have developed a simple theory on this basis and have theoretically estimated the efficiency of on-section labeling and compared it with experimental data. For randomly dispersed proteins in cytoplasm, Lowicryl sections will yield significant label only when the concentration of the antigen is about 10 microM or more. The complex situation of more compact proteins, as represented by fibers, sheets, and biological membranes is discussed and the difficulty of significant calculations is explained. Pre-embedding labeling and melted cryosections should give 10-30 times more label. The possible reasons for the observed much smaller gain of not more than two to three times are discussed.