Sulfide quinone reductase (SQR) activity in Chlorobium.

Membranes of the green sulfur bacterium, Chlorobium limicola f. thiosulfatophilum, catalyze the reduction of externally added isoprenoid quinones by sulfide. This activity is highly sensitive to stigmatellin and aurachins. It is also inhibited by 2-n-nonyl-4-hydroxyquinoline-N-oxide, antimycin, myxothiazol and cyanide. It is concluded that in sulfide oxidizing bacteria like Chlorobium, sulfide oxidation involves a sulfide-quinone reductase (SQR) similar to the one found in Oscilatoria limnetica [Arieli, B., Padan, E. and Shahak, Y. (1991) J. Biol. Chem. 266, 104-111].     

Campylobacter fetus Subspecies Contain Conserved Type IV Secretion Systems on Multiple Genomic Islands and Plasmids

The features contributing to differences in pathogenicity of the Campylobacter fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode a type IV secretion system (T4SS) and fic domain (filamentation induced by cyclic AMP) proteins, which may disrupt host cell processes. In the genomes of 27 C. fetus strains, three phylogenetically-different T4SS-encoding regions (T4SSs) were identified: one was located in both the chromosome and in extra-chromosomal plasmids; one was located exclusively in the chromosome; and one exclusively in extra-chromosomal plasmids. We observed that C. fetus strains can contain multiple T4SSs and that homologous T4SSs can be present both in chromosomal genomic islands (GI) and on plasmids in the C. fetus strains. The GIs of the chromosomally located T4SS differed mainly by the presence of fic genes, insertion sequence elements and phage-related or hypothetical proteins. Comparative analysis showed that T4SS sequences, inserted in the same locations, were conserved in the studied C. fetus genomes. Using phylogenetic analysis of the T4SSs, it was shown that C. fetus may have acquired the T4SS regions from other Campylobacter species by horizontal gene transfer. The identified T4SSs and fic genes were found in Cff and Cfv strains, although the presence of T4SSs and fic genes were significantly associated with Cfv strains. The T4SSs and fic genes could not be associated with S-layer serotypes or geographical origin of the strains.     

Assessing oligomerization of membrane proteins by four-pulse DEER: pH-dependent dimerization of NhaA Na+/H+ antiporter of E. coli

The pH dependence of the structure of the main Na(+)/H(+) antiporter NhaA of Escherichia coli is studied by continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) techniques on singly spin-labeled mutants. Residues 225 and 254 were selected for site-directed spin labeling, as previous work suggested that they are situated in domains undergoing pH-dependent structural changes. A well-defined distance of 4.4 nm between residues H225R1 in neighboring molecules is detected by a modulation in double electron-electron resonance data. This indicates that NhaA exists as a dimer, as previously suggested by a low-resolution electron density map and cross-linking experiments. The modulation depth decreases reversibly when pH is decreased from 8 to 5.8. A quantitative analysis suggests a dimerization equilibrium, which depends moderately on pH. Furthermore, the mobility and polarity of the environment of a spin label attached to residue 225 change only slightly with changing pH, while no other changes are detected by CW EPR. As antiporter activity of NhaA changes drastically in the studied pH range, residues 225 and 254 are probably located not in the sensor or ion translocation sites themselves but in domains that convey the signal from the pH sensor to the translocation site.     

Proteinases encapsulated in stimulated release liposomes for cheese ripening

Chymosin, a neutral proteinase from Bacillus subtilis and cardoon cyprosins, were co-encapsulated with phospholipase C in stimulated release liposomes. Encapsulated enzymes were added separately to milk to make cheese. Chymosin and the neutral proteinase accelerated as-casein degradation in comparison with control cheese, whereas b-casein degradation was accelerated by neutral proteinase and cyprosins. Neutral proteinase yielded the highest increase in soluble nitrogen. Cheese flavour intensity was enhanced by the neutral proteinase and cyprosins but not by chymosin.     

The proton electrochemical gradient in Escherichia coli cells.

The internal pH of Escherichia coli cells was estimated from the distribution of either 5,5-[14C]dimethyl-2,4-oxazolidinedione or [14C]methylamine. EDTA/valinomycin treatment of cells was employed to estimate delta psi from 86Rb+ distribution concomitant with the delta pH for calculation of delta muH. Respiring intact cells maintained an internal pH more alkaline by 0.63-0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The delta pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The delta muH, established by EDTA/valinomycin-treated cells, was constant (122-129 mV) over extracellular potassium concentration of 0.01 mM-1 mM. At the lower potassium concentration delta psi (110-120 mV) was the predominant component, and at the higher concentration delta pH increased to 0.7 units (42 mV). At 150 mM potassium delta muH was reduced to 70 mV mostly due to a delta pH component of 0.89 (53 mV). The interchangeability of the delta muH components is consistent with an electronic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of delta muH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone. The highest delta pH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the delta pH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of delta pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the delta psi was almost constant over the whole range of extracellular pH values (6-8) implying electroneutral proton movement. Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.     

Polarizability of neutral copper clusters

A theoretical study of the electric-field effect on the electronic structures and related properties of the cation compound containing 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and imidazole unit has been carried out, using the density functional theory (DFT) at the (U) B3LYP/6-31+G(d,p) level. The changes and regularities of geometric and electronic properties of the researched compound under electric field were revealed in detail. The results show the following: (1) Electric field has a very important effect on the orbital energy, dipole moment, natural population, and structure of the cation compound. Most of these properties are changed orderly with the increase of the electric-field intensity. (2) It is very interesting to find that in the present different electric-field intensities, the structure of cation compound after getting an electron becomes bis-radical form, that is, no mater in or out of electric-field, the cation compound will exist in a triplet state after getting an electron. (3) When getting an electron, the change of the cation structure mainly appears on the imizadole head, and when losing an electron, the change mainly appears on the TEMPO head. These theoretical results considering the electric-field effect for the cation compound help to explain the related experimental phenomena and further to direct the functional molecular design of this kind of compound.     

Expression patterns of BMPRs in the developing mouse molar

During development, Bone Morphogenetic Proteins (BMPs) can induce apoptosis, cell growth or differentiation. These different effects are mediated by dimers of two types of BMP–receptors (BMPRs). To identify the responding cells during tooth development and search for possible tissue–or stage–specificities in the receptors involved, the distribution patterns of BMPR–IA, –IB and –II were investigated in the mouse molar, from bud to bell stage. At the bud stage, BMP–2 was suggested to be involved in the formation of an epithelial signaling center, the primary enamel knot (PEK), while BMP–4 would mediate the condensation of the mesenchyme. Immunostaining showed the presence of BMPR–IA and –II in the epithelium instead of BMPR–IB and –II in the mesenchyme. At the cap stage, BMPR–IB was detected in the epithelium but not BMPR–II, suggesting the existence of another type II receptor to form a functional dimer. At the late cap stage in the epithelium, BMP–4, BMPR–IA and –II were restricted to the internal part of the PEK and the stalk: two apoptotic areas. The three proteins were detected in the mesenchyme, showing a strong staining where cusps were about to form. At the late bell stage, BMP–2 or –4 may induce cell differentiation. BMPR–IB and –II were detected in odontoblasts instead of BMPR–IA and –II in ameloblasts. These results provide the first evidence of multiple type I and type II BMP–receptors, expressed in the dental epithelium and mesenchyme at different stages of development, to signal different cellular activities in a time– and tissue–specific way.     

Generation of a human IgM monoclonal antibody directed against HLA class II molecules: a potential agent in the treatment of haematological malignancies

Major histocompatibility complex (MHC) class II molecules have been considered as a good target molecule for use in immunotherapy, because of the high expression in some lymphoma and leukaemia cells and, also, because of their restricted expression on human cells (monocytes, dendritic, B lymphocytes, thymic epithelial cells, and some cytokine-activated cells, such as T lymphocytes). We have obtained a human IgM monoclonal antibody directed against human leukocyte antigen (HLA) class II molecules, using transgenic mice carrying human Ig genes. The antibody BH1 (IgM/κ isotype) recognises HLA-class II on the surface of tumour cells from patients suffering from haematological malignancies, such as chronic and acute lymphocytic leukaemias, non-Hodgkin lymphomas and myeloid leukaemias. Interestingly, functional studies revealed that BH1 mAb recognises and kills very efficiently tumour cells from several leukaemia patients in the presence of human serum as a source of complement. These results suggest that this human IgM monoclonal antibody against HLA-class II could be considered as a potential agent in the treatment of several malignancies. Belén Díaz, Irene Sanjuan and Susana Magadán share authorship; Francisco Gambón and áfrica González–Fernández share leadership.