Site-directed mutagenesis to determine essential residues of ribulose-bisphosphate carboxylase ofRhodospirillum rubrum

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.     

Interleukin 2 up-regulates its own production

It has been previously reported that a combination pair of anti-CD2 monoclonal antibodies (mAb) T11(2)+T11(3) induces a strong proliferation of T cells, which does not require the involvement of accessory cells and exogenous interleukin 2 (IL-2). More recently, we have shown that the requirement for optimal T cell proliferation depends on the combination pairs of anti-CD2 mAb used. Among them, anti-GT2+T11(1) mAb do not allow optimal proliferation of TA4 helper cloned T cells due, at least in part, to a low level of IL-2 production. This observation offered us the opportunity to study the effect of IL-2 on its own production. We show here that stimulation of cloned TA4 cells with anti-GT2+T11(1) mAb induces only a marginal level of IL-2 production. By contrast, significantly higher levels of IL-2 activity are detected in the culture supernatant of TA4 cells preincubated with recombinant IL-2 (rIL-2) before stimulation with anti-GT2+T11(1) mAb. This effect is dose-dependent over a wide range (5 to 50 IU/ml) of rIL-2 concentrations added during preincubation time. In addition, it is not due to carryover of rIL-2 bound during the preincubation time, or to lesser IL-2 consumption by these cells, or to increasing numbers of IL-2-producing cells induced by exogenous IL-2. Moreover, the observation was confirmed with IL-2 mRNA. Although neither rIL-2 nor anti-GT2+T11(1) mAb alone could induce a significant production of IL-2, rIL-2 appears to up-regulate its own production when the TA4 cells are activated by the anti-CD2 mAb-mediated second signal.     

Population Heterogeneity of Agrobacterium tumefaciens in Galls of Populus L. from a Single Nursery

This study focused on the natural crown gall infections occurring in a Leuce poplar nursery. Soil effects on crown gall frequency were detected, indicating that contamination was due to a resident Agrobacterium tumefaciens population, which was present before seedling plantation. The crown gall frequency on poplar progenies varied from 3 to 67%, indicating the feasibility of improvement in crown gall resistance. Of 129 tumor isolates, 128 were pathogenic. These isolates were of biotype 1 or 2. Biochemical, serological, and antibiotic resistance typing results concurred, indicating the presence of four biotype 1 and two biotype 2 resident subpopulations. No significant change was noticed in the relative proportions of subpopulations from one year to another. Pathogenic subpopulations both in vitro and in planta were susceptible to Kerr K84 (P. B. New and A. Kerr, J. Appl. Bacteriol. 90:172-179, 1972). In addition, no serological cross-reactions were found to occur between K84 and the pathogenic subpopulations.     

Compatability of Metarhizium anisopliae var. anisopliae with chemical pesticides

The effects of various insecticides on the mycelial growth, sporulation and conidial germination of Metarhizium anisopliae var. anisopliae isolate E₉ were studied in the laboratory. Chlorpyrifos was the most toxic organophosphate to mycelial growth and sporulation at all concentrations. Temephos, malathion and leptophos were highly toxic to sporulation while malathion was the most inhibitory to germination. The carbamates, carbofuran, methomyl and oxamyl were moderately toxic to mycelial growth and sporulation while oxamyl had an adverse effect on germination. The pyrethroids (pyrethrin, permethrin and resmethrin) and the insect growth regulators (diflubenzuron and methoprene) were not inhibitory to the various developmental stages of isolate E₉. The chlorinated hydrocarbons (chlordane, lindane and toxaphene) were more deleterious than all other insecticide groups tested. Among the fungicides, benomyl and maneb produced the greatest inhibition.     

ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed thatS-adenosylmethionine: protein-lysineN-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2,3-cyclic nucleotide 3-phosphohydrolase and 5-nucleotidase as well asS-adenosyl-methionine: protein-carboxylO-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.     

Production of Vero cytotoxin by strains of Escherichia coli as related to the availability of iron

Abstract Vero cytotoxin (VT) producing strains of Escherichia coli (VTEC), including isolates from cases of haemolytic uraemic syndrome and infantile diarrhoea, were used to determine the effect of iron availability on the production of intra- and extracellular VT, with particular interest in elevating toxin production by low-level toxin producing VTEC. Culturing bacteria under iron restriction resulted in growth retardation and a decrease in the production of VT. For the routine detection of both high- and low-level VT-producing E. coli , there was no advantage to be gained by growing bacteria under iron restriction or using disrupted bacterial cell preparations; on the contrary, testing culture supernatants from bacteria grown in iron-replete media for approximately 14 h proved to be the most sensitive and accurate method for detecting VT and the resultant identification of VTEC.     

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type