Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation

A synthetic gene coding for the cysteine proteinase inhibitor (desSer1 Ile29 Leu89) chicken cystatin was cloned and expressed in E. coli. The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8. Expression as fusion protein was performed in a temperature-inducible E. coli system. The expression product was synthesized as 20% of total E. coli protein. The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N-terminal sequence confirmed up to position 37. The properties of the purified material correspond to those of natural chicken cystatin. The recombinant cystatin variant binds anti-chicken cystatin IgG, is inhibitorily active and displays Ki values with papain and with cathepsin B similar to those determined for natural chicken cystatin.     

Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.     

Characterization of a newin vitro model for studies of reepithelialization in human partial thickness wounds

Summary Reepithelialization of artificial partial thickness wounds made in biopsies of human skin was determined after 3, 5, or 7 d of incubation, submerged or elevated to the air-liquid interface. The biopsies were reepithelialized within 5–7 d, with a more complete epidermal healing in wounds exposed to air. Both types of wounds showed similar time-course in deposition of basement membrane components, as detected by immunofluorescence labeling. Laminin and collagen type VII were deposited underneath the migrating tips, whereas collagen type IV was detected after reepithelialization. Markers of terminal differentiation showed a pattern close to normal in the air-liquid incubated wounds after reepithelialization. Involucrin was detected in the suprabasal regions of the migrating epidermis and thereafter in the upper half of neo-epidermis in the air-liquid incubated wound. Filaggrin could not be detected in the submerged wounds at any time during healing, whereas wounds exposed to air showed a well-differentiated epidermis by Day 7. Tritiated thymidine-incorporation indicated proliferation of epidermal and dermal cells during reepithelialization and a maintained viability, as shown by cultivation of endothelial- and fibroblast-like cells obtained from the dermis 7 d after wounding. Reepithelialization in this humanin vitro model is supported by a matrix close to normal with the possibility of extracellular influences and cell-cell interactions and, in addition, the technique is simple and reproducible. Therefore, we suggest this model for studies of regeneration in culture and as a complement toin vivo studies on epidermal healing.     

Male-specific cell migration into the developing gonad

Background: The gene Sry acts as a developmental switch, initiating a pathway of gene activity that leads to the differentiation of testis rather than ovary from the indifferent gonad (genital ridge) in mammalian embryos. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. To investigate the contribution of mesonephric cells to this process, gonads from wild-type mice (CD1), and mesonephroi from a transgenic strain ubiquitously expressing β-galactosidase (ROSA26), were grafted together in vitro. After culture, organs were fixed and stained for β-galactosidase activity to identify cells contributed from the mesonephros to the male or female gonad.Results: Migration of mesonephric cells occurred into XY but not XX gonads from 11.5–16.5 days post coitum (dpc). Somatic cells contributed from the mesonephros were distinguished by their histological location and by available cell-specific markers. Some of the migrating cells were endothelial; a second population occupied positions circumscribing areas of condensing Sertoli cells; and a third population lay in close apposition to endothelial cells.Conclusions: Migration from the mesonephros to the gonad is male specific at this stage of development and depends on an active signal that requires the presence of a Y chromosome in the gonad. The signals that trigger migration operate over considerable distances and behave as chemoattractants. We suggest that migration of cells into the bipotential gonad may have a critical role in initiating the divergence of development towards the testis pathway.     

Citric acid production in chemically defined media by Aspergillus niger

Summary The effect of changing the composition of a chemically defined medium on citric acid production by Aspergillus niger was investigated. High and reproducible amounts of citric acid were obtained with deionized commercial sugar solutions, proper phosphate concentrations, low initial pH values and suitable amounts of copper as growth inhibiting agent.Comparison of high and low yielding process parameters showed that under high yielding conditions, (deionized sugar, Cu⁺⁺ addition) besides more citric acid, less mycelium and less mycelial lipids were formed; the consumption of sugar, nitrogen and phosphorus was related to the amount of biomass.Partly presented at the XII. International Congress of Microbiology; München, September 3–8, 1978     

Wild-type Leishmania donovani promastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells--the influence of phosphoglycans.

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.     

In vitro compatibility of Beauveria bassiana strain ATCC 74040 with copper fungicides

Copper fungicides and mycoinsecticides based on entomopathogenic fungi Beauveria spp. are the most common pesticides used in organic crop production systems. The in vitro effects of the copper fungicides copper oxide, copper hydroxide, copper oxychloride, copper sulphate, dicopper chloride trihydroxide and tribasic copper sulphate were investigated for mycelial growth, sporulation and conidial germination of the ATCC 74040 commercial strain of Beauveria bassiana. Mycelial growth was evaluated on potato dextrose agar plates with 100%, 75%, 50%, 25%, 12.5%, 6.25% and 0% of the recommended application rates of each fungicide at 15 and 25°C. Sporulation and conidial germination were determined at the recommended field doses of each fungicide at 25°C. All copper fungicides had fungistatic or fungicidal effects on B. bassiana that varied according to the dose. Only in two cases, copper oxide at 15°C and copper hydroxide at 25°C, at the lowest concentration of 6.5%, was mycelial growth not statistically significantly inhibited. Inhibition of mycelial growth depended both on the fungicide and its concentration, and partly on temperature. Both sporulation and conidial germination of B. bassiana were significantly inhibited by all fungicides. All fungicides inhibited the sporulation in a similar way (99.8%–100%). With the exception of copper oxychloride (inhibition, 13.6%), the other fungicides showed high detrimental effects on conidial germination (inhibition, 91.7%–100%). The fungus was strongly affected by some fungicides even at the lowest doses. The biological index used for the B. bassiana with copper fungicides ranged from 0.6 (copper sulphate) to 18.1 (copper oxychloride). Therefore, the tested fungicides were classified into the upper half of the highly toxic (T) category and are considered incompatible with the entomopathogenic fungus B. bassiana strain ATCC 74040 under in vivo experimental conditions. These results need to be further verified in vitro under both greenhouse and open-field conditions.