The membrane topology of proton-pumping Escherichia coli transhydrogenase determined by cysteine labeling.

The membrane topology of proton-pumping nicotinamide-nucleotide transhydrogenase from Escherichia coli was determined by site-specific chemical labeling. A His-tagged cysteine-free transhydrogenase was used to introduce unique cysteines in positions corresponding to potential membrane loops. The cysteines were reacted with fluorescent reagents, fluorescein 5-maleimide or 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid, in both intact cells and inside-out vesicles. Labeled transhydrogenase was purified with a small-scale procedure using a metal affinity resin, and the amount of labeling was measured as fluorescence on UV-illuminated acrylamide gels. The difference in labeling between intact cells and inside-out vesicles was used to discriminate between a periplasmic and a cytosolic location of the residues. The membrane region was found to be composed of 13 helices (four in the alpha-subunit and nine in the beta-subunit), with the C terminus of the alpha-subunit and the N terminus of the beta-subunit facing the cytosolic and periplasmic sides, respectively. These results differ from previous models with regard to both number of helices and the relative location and orientation of certain helices. This study constitutes the first in which all transmembrane segments of transhydrogenase have been experimentally determined and provides an explanation for the different topologies of the mitochondrial and E. coli transhydrogenases.     

A bacterial clone carrying sequences coding for elongation factor EF-1 alpha from Artemia

A bacterial cDNA clone was identified carrying one third of the nucleotides coding for elongation factor EF-1 alpha from the brine shrimp Artemia. The sequence of codons corresponds with the known sequence of amino acids of EF-1 alpha in the region involved.     

A new semi-automated instrument to improve the fine needle aspiration procedure during breast lesion cell sampling

A large and increasing number of women in the western world will at some point during their life be investigated morphologically for some type of breast lesion. Fine Needle Aspiration (FNA) is one morphological method which is considered to be the fastest, cheapest and the most patient-friendly approach. However, the frequency of conclusive samples using this method varies and is often too low, especially when performed by unexperienced operators. In this study we have developed and tested a new semi-automated instrument (“CytoTest”) designed for FNA which is intended to improve the efficacy of the technique by increasing the percentage of conclusive samples. A total of 443 consecutive aspiration procedures on palpable breast lesions were performed to compare this new “CytoTest” equipment with the standard protocol using the same type of needles. We conclude that by increasing the extent and frequency of the reciprocatory motions used by an experienced sampling operator as well as enhancing the ejection pressure, the cellular yield can be increased almost three folded compared to the standard protocol. For cases with high amounts of non-diagnostic material (such as blood or cystic fluid) which were discarded, up to four times more sample could be obtained. Furthermore, the frequency of sparse samples under 1 mg was halved with use of the “CytoTest”.     

Plant Reactions to Inoculation of Roots with Fungi and Bacteria

The potential of 120 isolates of fungi and bacteria from plant rhizospheres to interfere with plant development and growth was studied in greenhouse experiments. The pure cultured isolates were obtained from plant roots in the field and applied as suspensions to the roots of eight test plant species. 10–20% of the isolates caused distinct symptoms on shoots, growth retardations without other symptoms or growth promotions. Responses of treated plants ranged from death of plants soon after treatment to up to about 40% higher shoot fresh weight than in control plants. Two bacterial isolates induced strong reactions in most of the plant species tested while other isolates showed a more or less pronounced specificity by giving reactions in only some of the plant species tested.     

Role of antigen and,antibody in the regulation of the immune response

The enzyme dextranase could degrade antigenic dextran in vivo even when given 6-15 d after the antigen. Dextranase injected after the antigen suppressed the immune response when given 24 but not 48 h after the antigen, indicating that the antigen must interact with the immune system for 48 h to initiate a response. Thereafter, the B cells are independent of further antigen stimulation. To show whether antibody-mediated suppression of the immune response was determinant specific FITC-conjugated SRC were applied as immunogen and antibodies were raised both against the carrier (SRC) and the FITC hapten. When these antibodies were injected 1-3 h after the immunogen they only suppressed the immune response to the corresponding determinant. Anti-carrier antibodies usually enhanced the response to the hapten. Therefore, antibody-mediated suppression of the immune response is determinant-specific and cannot be mediated in vivo to a detectable extent by the Fc part of the antibodies.     

Pulsed-field gel electrophoresis for genotypic comparison of Rhizobium bacteria that nodulate leguminous trees

Abstract Pulsed-field gel electrophoresis (PFGE) was applied to characterize Rhizobium bacteria isolated from the root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Keya. For the electrophoresis, the total DNA of 42 isolates, embedded in agarose, was digested by a rare-cutting restriction endonuclease, Xba I. The PFGE run resulted in good resolution of the DNA fragments and gave the strains distinctive fingerprint patterns. The patterns were analysed visually and using automated clustering analysis, which divided the strains into groups resembling the results generated by numerical taxonomy. However, several strains had unique banding patterns, which indicates that these strains are genetically very diverse.     

The chemical-transformation products of 1,6-dibromo-1,6-dideoxygalactitol and 1,2:5,6-dianhydrogalactitol in aqueous solution

After hydrolysis of 1,6-dibromo-1,6-dideoxygalactitol (1) and 1,2:5,6-dianhydrogalactitol (2), 11 compounds were isolated, three of them as tritylated derivatives. Their structures were established on the basis of chemical evidence and, for four compounds, by X-ray diffraction. The main product of the hydrolysis of 1 was 3,6-anhydro-1-bromo-1-deoxy-dl-galactitol; the end-products of the hydrolysis of 2 were 1,5-anhydro-dl-galactitol, 2,5-anhydro-dl-altritol, and galactitol.