Litter parameters and spontaneous abnormalities in Himalayan rabbits

A joint study was undertaken in three testing facilities to evaluate cumulative background data of Himalayan rabbits. All litters were derived from control does. The conception rate was high (84.0-95.1%) but the average numbers of corpora lutea (7.9-8.7), implantation sites (6.5-7.5) and viable fetuses (5.8-6.9) were somewhat lower than that of most other strains of rabbit. Altogether 90 malformed fetuses (1.12%) and 425 fetuses with variations (5.27%) occurred among 8,060 virable fetuses.     

Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose

The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin.     

The consensus concept for thermostability engineering of proteins: further proof of concept

Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.     

An automatic system for the assessment of complex medium additives under cultivation conditions

Complex medium additives such as yeast extract or peptone are often used in industrial cell culture processes to prolong cell growth and/or to improve product formation. The quality of those supplements is dependent on the preparation method and can differ from lot to lot. To guarantee consistent production these different lots have to be tested prior to use in fermentation processes. Because a detailed qualitative and quantitative analysis of all components of such a complex mixture is a very difficult task, another assessment method has to be chosen. The best way to evaluate the effect of such supplements is to monitor cell activity during real cultivation conditions with and without the added supplement lot. A bioreactor-based test system has been developed to determine the oxygen requirement of the cells as a response to the addition of a supplement to be tested under standardized conditions. Investigations were performed with a mouse-mouse hybridoma cell line and yeast extracts as an example for complex medium additives. The results showed differences in the impact between different extract lots and between different concentrations of an extract.     

Species-specific probes and real-time PCR as a tool for fast detection and differentiation of 15 bacteria relevant in intensive care medicine

Detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and blood has the potential to improve diagnostic tools in microbiology. A novel real-time PCR-based assay was developed and its performance and robustness were evaluated for a panel of spiked suspensions of 15 clinically relevant bacteria. As low as ten colony forming units (CFU)/100 μl were detectable. No cross-reactivity was observed, except for Staphylococcus aureus and Staphylococcus epidermidis. Nevertheless, S. aureus and S. epidermidis were reliably differentiated by melting curve analysis. The protocol was also validated with three groups containing a mixture of five spiked bacteria each, with the result of reliable differentiation. This novel assay allows an exact identification of 15 microbes relevant in intensive care medicine, including mixed infections, in a one run experiment in less than 4 h.     

The myoglobin of primates. VIII. Gorilla gorilla beringei (eastern highland gorilla).

On aligning the tryptic peptides of the myoglobin from a gorilla with the homologous human peptides, one amino acid difference was found. By dansyl-Edman degradation this was shown to be at position 22, i.e. at a position other than those where man, chimpanzee and gibbon differ from one another.     

Quantitative field desorption mass spectrometry. V. Discussion of methodology and examples of applications

The possibilities for the application of field desorption mass spectrometry in quantitative analyses are described and evaluated. The advantages of and the sources of errors in the use of different standards as well as in the application of different methods such as photographic detection, single ion monitoring, repetitive scanning, selected ion monitoring, and double ion detection are illustrated by representative examples. Sensitivity and precision of the different techniques are evaluated. Most importantly, the use of stable isotope labelled compounds as internal standards has enabled quantitative determination with good precision, accuracy, and sensitivity. In order to demonstrate the capabilities of the methods, examples of applications are presented and the scope of quantitative analysis with field desorption mass spectrometry is discussed.     

3-Azi-1-methoxybutyl D-maltooligosaccharides specifically bind to the maltose/maltooligosaccharide-binding protein of Escherichia coli and can be used as photoaffinity labels

Maltooligosaccharides with two to six (alpha 1-4)-linked glucose residues, carrying at their reducing end a 3-azi-1-methoxybutyl group in either alpha or in beta glycosidic linkage, were synthesized. These maltooligosaccharide analogues inhibit maltose uptake via the maltose-binding-protein-dependent transport system in Escherichia coli. The concentration of half-maximal inhibition of maltose transport, at 15 nM concentration, decreases with increasing chain length of the analogue, levelling off at 40 microM after a chain length of four glucose residues in the alpha series and at 350 microM after a chain length of three glucose residues in the beta series. The inhibition of maltose transport occurs at the level of the periplasmic maltose-binding protein. 3-Azi-1-methoxybutyl alpha-D-[3H]maltotrioside was bound by the maltose-binding protein with a Kd of 0.18 mM. Irradiation at 350 nm of purified maltose-binding protein in the presence of 4 microM of this substrate labeled the protein covalently; labeling was prevented by 1 mM maltose. Using a crude preparation of periplasmic proteins two proteins were labeled, the maltose-binding protein and alpha-amylase. Thus, 3-azi-1-methoxybutyl alpha-D-maltooligosaccharides are potent photoaffinity labels for proteins with maltooligosaccharides-binding sites.