Sex-dependent effects of non-H-2 loci on lethal GVH reaction induced across an H-2 barrier

We have studied the influence of DBA/2 non-H-2 antigens on the lethal graft-versus-host reaction (GVHR) developed across an H-2 barrier. (DBA/2 x B10.D2)F₁ x B10.D2 (H-2 ^d) backcross (BC) mice were typed for their allelic constitution at nine genetically independent chromosome markers and used as individual cell donors simultaneously for two to three (DBA/2 X B10.D2)F₁ recipients incompatible for DBA/2 non-H-2 antigens alone and two to three (DBA/2 x B10.BR)F₁ recipients incompatible for DBA/2 non-H-2 antigens and H-2^k. The results showed that, when compared with that developed in a control group incompatible for H-2 ^kalone [B10.D2(B10.D2xB10.BR)F₁], the GVHR mortality seen in the presence of an additional incompatibility for DBA/2 non-H-2 antigens [(DBA/2 X B10.BR)F₁recipients] is significantly delayed but only in female mice. An analysis of individual BC donors indicated that this protective effect of DBA/2 non-H-2 antigens correlates with incompatibility for gene(s) linked to the Pgm-1 chromosome marker. In contrast, incompatibility for gene(s) linked to Mod-1 and Es-3 markers accelerates GVHR mortality, but only in male mice. Finally, the results obtained with (DBA/2 x B10.D2)F₁ and (DBA/2 x B10.BR)F₁ recipients were compared; they showed that the intensity of the GVHR developed by cells from individual BC donors against a given set of DBA/2 non-H-2 antigens correlates well with that developed by the same BC donor against the same set of non-H-2 antigens plus H-2^k. We conclude that certain non-H-2 genes (and antigens) can modulate the intensity of the GVHR developed across an H-2 barrier. The number of such genes is probably great; their effects are strong and complex, and can be sex-dependent.     

Characterization and expression of collagen-like genes in Drosophila melanogaster

We have used a cloned chicken collagen cDNA sequence to help identify hypothetic members of the collagen gene family from Drosophila melanogaster. Several experimental evidences have been obtained which indicate that the Drosophila genome contains numerous collagen-like sequences. We have characterized in more detail ten distinct DNA sequences that hybridized strongly to the heterologous collagen probe. By in situ hybridization we have shown that these sequences are dispersed throughout the Drosophila genome. Two of them are shown to originate from the previously described DCg 1 and DCg 2 collagen genes. In other respects, we show that in addition to DCg 1 and DCg 2, at least five putative collagen genes are expressed during the Drosophila lifetime. These genes are unique, and some of them are seen to be transcribed into different size classes of mRNAs. Additionally, the data presented so far demonstrate that the expression of these genes is regulated temporally and/or quantitatively during the Drosophila life cycle.     

Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells

Summary Chromaffin granules from bovine adrenal medullary chromaffin cells have been found to contain small vesicular structures bounded by unit membranes. Detection of these intragranular vesicles within intact cells requires the use of quick-freezing methods. The intragranular vesicles are labile to fixation by aldehydes which explains why they have not been described in intact cells until now. They are found in approximately 60% of the dense-core chromaffin granules in cells and 85% of isolated granules. They are usually clustered in groups of one to as many as five between the core and the inner surface of the granule membrane. The intragranular vesicles are independent vesicles in that they do not appear as simple invaginations of the granule membrane in either serial thin-section or freeze-etch views. Furthermore, they are released from the cell along with granule contents during nicotine-induced secretion of catecholamines. The structural heterogeneity provided by the intragranular vesicles may be related to the functional heterogeneity of granule contents observed in many recent biochemical studies.     

Variations in the response of salt-stressed Rhizobium strains to betaines

A total of 15 rhizobial strains representing Rhizobium meliloti, Rhizobium japonicum, Rhizobium trifolii, Rhizobium leguminosarum, Rhizobium sp. (Sesbania rostrata) and Rhizobium sp. (Hedysarum coronarium), were studied with regard to growth rate under salt stress in defined liquid media. In the presence of inhibitory concentrations of NaCl, enhancement of growth resulting from added glycine betaine was observed for R. meliloti strains and Rhizobium sp. (Hedysarum coronarium) but not for other Rhizobium species. The concentration of glycine betaine required for maximal growth stimulation was very low (1 mM) in comparison with the osmolarity of the medium. The stimulation was shown to be independent of any specific solutes. Other related compounds like proline betaine, carnitine, choline, -butyrobetaine and pipecolate betaine were also effective compounds in restoring the growth rate of cells grown in medium of elevated osmolarity. High rate of glycine betaine uptake was demonstrated in R. meliloti cells grown in media of increased osmotic strength. The intracellular concentration of this solute was found to be 308 mM in 0.3 M NaCl-grown cells and 17 times lower in minimal medium-grown cells. Glycine betaine was used for growth under conditions of low osmolarity but could not serve as sole carbon or nitrogen source in medium of increased osmotic strength. Experiments with [¹⁴C]glycine betaine showed that this molecule was not metabolized by cells subjected to osmotic stress, whereas it was rapidly converted to dimethylglycine, sarcosine and glycine in minimal medium-grown cells.Abbreviations LAS lactate-aspartate-salts - LGS lactate-glutamate-salts - LS lactate-succinate - MSY mannitol-salts-yeast - YLS yeast-lactate-succinate     

Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli.

A clone bearing the structural gene sacB, coding for the exoenzyme levansucrase, was isolated from a library of Bacillus subtilis DNA that was cloned in phage lambda charon 4A on the basis of the transforming activity of the chimeric DNA. This lambda clone also was found to contain the sacR and smo loci. Subcloning the sacB-sacR region in plasmid pBR325 resulted in a clone which directed levansucrase synthesis in Escherichia coli. The nucleotide sequence coding for the secreted protein was localized on the physical map of the cloned DNA.     

Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes

Leishmania tropica, Trypanosoma brucei, Trypanosoma cruzi, and Crithidia fasciculata have superoxide dismutases which are insensitive to cyanide and sensitive to peroxide and azide, properties characteristic of iron-containing superoxide dismutase. Studies on the superoxide dismutase of C. fasciculata have revealed that: 1) the enzyme is located in the cytosol; 2) isozymes exist; 3) the major superoxide dismutase isozyme (superoxide dismutase 2) has Mr approximately equal to 43,000 and consists of two equal-sized subunits, each of which contains 1.4 atoms of iron. Comparisons of the amino acid content of this crithidial superoxide dismutase with those of superoxide dismutases from other sources suggests that the crithidial enzyme is closely related to bacterial Fe-containing superoxide dismutases, and only distantly related to human Mn- and Cu,Zn-containing superoxide dismutases and to Euglena Fe-containing superoxide dismutase. Attempts are now underway to develop specific inhibitors of the trypanosomatid superoxide dismutase which may be of use in the treatment of leishmaniasis or trypanosomiasis.     

Mode of interaction of polyoxyethyleneglycol detergents with membrane proteins

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.     

Kinetic studies of aminoglycoside acetyltransferase and phosphotransferase from Staphylococcus aureus RPAL. Relationship between the two activities

In the Staphylococcus aureus strain harbouring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from two inactivating reactions catalyzed by a 6'-N-aminoglycoside acetyltransferase and a 2"-O-amino-glycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography. The kinetic mechanisms of each activity have been determined from studies of initial velocities, as well as product and dead-end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of one reaction have been tested as effectors of the other reaction. No interaction between the two activities has been observed. However, the GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltransferase against thermal inactivation, which suggests that the two activities may be associated.     

Plasmodium falciparum: Drug sensitivity in vitro of isolates before and after adaptation to continuous culture

Fifteen strains of Plasmodium falciparum have been cultivated since 1979 using the Trager and Jensen method of continuous culture on isolates from malaria patients. One hundred and two drug sensitivity studies have been carried out on these strains using a semimicro test. Three isolates, initially resistant to chloroquine, adapted rapidly to in vitro cultivation and maintained their high level of resistance (ED₅₀ above 660 nM). Eleven isolates, initially chloroquine sensitive (ED₅₀ under 90 nM) became resistant to this drug (ED₅₀ = 190 to 1950 nM) after the 2–15 weeks required for their adaptation to continuous culture. The resistance of these strains never decreased during the following 15 months of continuous culture. The sensitivity to quinine varied initially from one strain to another (ED₅₀= 160 to 660 nM) and fluctuated during cultivation in the ratio of 1, 3.5 for a given strain. The sensitivity of mefloquine remained high for all strains (ED₅₀ under 150 nM) but one (ED₅₀ = 560 nM). These results suggest that there might be a relationship between in vitro adaptation to culture of P. falciparum by the Trager-Jensen method and a chloroquine-resistant characteristic of the strain. There is the possibility of the emergence of a drug-resistant subpopulation or of changes in the metabolic pathways.