Diversity among Rhizobia Effective with Robinia pseudoacacia L.

The diversity of rhizobia that form symbioses with roots of black locust (Robinia pseudoacacia L.), an economically important leguminous tree species, was examined by inoculating seedling root zones with samples of soil collected from the United States, Canada, and China. Bacteria were isolated from nodules, subcultured, and verified to be rhizobia. The 186 isolates varied significantly in their resistance to antibiotics and NaCl, their growth on different carbohydrates, and their effect on the pH of culture media. Most isolates showed intermediate antibiotic resistance, the capacity to use numerous carbohydrates, and a neutral to acid pH response. Isolates had greater similarity within sampling locations than among sampling locations. The isolates were grouped by using numerical taxonomy techniques, and representative strains of 37 groups were selected. The mean generation times of these isolates ranged from 3 to 9 h, and the protein profile of each of the 37 isolates was unique. Nitrogen fixation, total nitrogen accumulation, and plant growth varied significantly among black locust seedlings inoculated with the representative isolates. We conclude that great variation exists among Rhizobium spp. that nodulate black locust, and selection of strains for efficiency of the symbiotic association appears possible.     

Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification.

Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus- and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Acholeplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, Lactobacillus, Bacillus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.     

Morphological and biochemical evidence for partial nuclear localization of annexin 1 in endothelial cells.

Using immunofluorescence, an affinity-purified anti-annexin-1 polyclonal antibody showed both cytoplasmic and nuclear staining, whereas antibodies against annexins 2, 5 and 6 labelled almost exclusively the cytoplasm of cultured endothelial cells. This was further confirmed by immunogold labelling and electron microscopy using a monoclonal antibody, annexin 1 being detected close to the plasma membrane, in the cytoplasm, as well as inside the nucleus. Finally, using immunoblotting, purified nuclei were shown to contain annexin 1, which was not removed by EDTA treatment. These data open some new perspectives in the understanding of annexin function, including possible involvement in nucleoskeleton dynamics and regulation of proliferation through cell signalling.     

A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators.

The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (KM) of the reaction, whereas the catalytic rate constant (kcat) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the KM values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The kcat however, was lower with "KHRR" t-PA than with wild-type t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The broad diversity of neutral and sialylated oligosaccharides derived from human salivary mucins.

Mucin glycopeptides were prepared from the salivary mucins of 20 healthy donors with blood group O. The carbohydrate chains of the high-molecular-weight mucins were released by alkaline borohydride treatment. Neutral and monosialylated oligosaccharide-alditols were purified by ion-exchange chromatography, gel filtration, and HPLC. The structures of the oligosaccharide-alditols were determined by high-resolution 1H-NMR spectroscopy in combination with fast atom bombardment mass spectrometry and methylation analysis. Thirty-seven oligosaccharide-alditols were characterized and illustrate the extreme diversity of the salivary mucins carbohydrate chains. This diversity might represent a mosaic of bacterial adhesion sites and be involved in the early events of the nonimmune defense of the oral cavity. Among these 37 oligosaccharide-alditols, 31 have not been previously described in human saliva and five of these are novel structures: [formula: see text]     

Conformational states of ribulosebisphosphate carboxylase and their interaction with chaperonin 60.

Conformational states of ribulosebisphosphate carboxylase (Rubisco) from Rhodospirillum rubrum were examined by far-UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-naphthalenesulfonate (ANS) binding. At pH 2 and low ionic strength (I = 0.01), Rubisco adopts an unfolded, monomeric conformation (UA1 state) as judged by far-UV CD and tryptophan fluorescence. As with other acid-unfolded proteins [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577], an intermediate conformation (A1 state) is observed at pH 2 and high ionic strength. The A1 state has an alpha-helical content equivalent to 64% of that present in the native dimer (N2 state). However, fluorescence measurements indicate that the tertiary structure of the A1 state is largely disordered. A site-directed mutant, K168E, which exists as a stable monomer [Mural, R. J., Soper, T. S., Larimer, F. W., & Hartman, F. C. (1990) J. Biol. Chem. 265, 6501-6505] was used to characterize the "native" monomer (N1 state). The far-UV CD spectra of the N1 and N2 states are almost identical, indicating a similar secondary structure content. However, the tertiary structure of the N1 state is less ordered than that of the N2 state. Nevertheless, when appropriately complemented in vitro, K168E forms an active heterodimer. Upon neutralization of acid-denatured Rubisco or dilution of guanidine hydrochloride-denatured Rubisco, unstable folding intermediates (I1 state) are rapidly formed. At concentrations at or below the "critical aggregation concentration" (CAC), the I1 state reverts spontaneously but slowly to the native states with high yield (greater than 65%). The CAC is temperature-dependent. At concentrations above the CAC, the I1 and the A1 states undergo irreversible aggregation. The commitment to aggregation is rapid [ef. Goldberg, M. E., Rudolph, R., & Jaenicke, R. (1991) Biochemistry 30, 2790-2797] and proceeds until the concentration of folding intermediate(s) has fallen to the CAC. In the presence of a molar excess of chaperonin 60 oligomers, the I1 state forms a stable binary complex. No stable binary complex between chaperonin 60 and the N1 state could be detected. Formation of the chaperonin 60-I1 binary complex arrests the spontaneous folding process. The I1 state becomes resistant to interaction with chaperonin 60 with kinetics indistinguishable from those associated with the appearance of the native states. In vitro complementation analysis indicated that the product of the chaperonin-facilitated process is monomeric.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 2. Site-directed mutagenesis of the xylose binding site.

Site-directed mutagenesis in the active site of xylose isomerase derived from Actinoplanes missouriensis is used to investigate the structural and functional role of specific residues. The mutagenesis work together with the crystallographic studies presented in detail in two accompanying papers adds significantly to the understanding of the catalytic mechanism of this enzyme. Changes caused by introduced mutations emphasize the correlation between substrate specificity and cation preference. Mutations in both His 220 and His 54 mainly affect the catalytic rate constant, with catalysis being severely reduced but not abolished, suggesting that both histidines are important, but not essential, for catalysis. Our results thus challenge the hypothesis that His 54 acts as an obligatory catalytic base for ring opening; this residue appears instead to be implicated in governing the anomeric specificity. With none of the active site histidines acting as a catalytic base, the role of the cations in catalyzing proton transfer is confirmed. In addition, Lys 183 appears to play a crucial part in the isomerization step, by assisting the proton shuttle. Other residues also are important but to a lesser extent. The conserved Lys 294 is indirectly involved in binding the activating cations. Among the active site aromatic residues, the tryptophans (16 and 137) play a role in maintaining the general architecture of the substrate binding site while the role of Phe 26 seems to be purely structural.     

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells.

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of SCP2 was present in the cytosol and that the distribution between membranes and cytosol was regulated by luteinizing hormone. The present data raise the question in what way SCP2 is involved in cholesterol transport between membranes in steroidogenic cells but also in non-steroidogenic cells.     

The interaction of tetanus toxin with intact bovine adrenal chromaffin cells: binding of toxin and subsequent inhibition of catecholamine release.

Tetanus toxin (about 1 nM) inhibits 70% of the nicotine-evoked release of catecholamines from intact adrenal medullary chromaffin cells after 20 h of incubation and 30% of the K(+)-evoked release. Inhibition of Ca(2+)-evoked release from detergent-permeabilized cells requires higher concentrations of toxin (about 1 microM) toxin, but is maximal after 12 min. Preincubation of the intact cells with ganglioside GT1 in the absence of toxin also inhibits evoked secretion. 125I-labelled toxin bound specifically to these cells; the binding capacity was greater at pH 6 (about 1 pmol toxin/mg cell protein) than at pH 7.4 (about 0.25 pmol). In both cases there were at least two binding components: one of high affinity (Kd about 1 nM) accounting for about 20% of total binding and one of lower affinity (Kd 10-20 nM). Preincubation of the cells with ganglioside increased the binding capacity, but did not affect the Kd of the lower affinity component. Similar observations could be made when binding was measured immunocytochemically. Extraction of gangliosides from chromaffin cells and overlay experiments with radiolabelled toxin showed that, as well as GM3, the major ganglioside component of chromaffin cell membranes, a ganglioside having the chromatographic mobility of GT1 was a major ligand for toxin.