Nuclear magnetic resonance investigation of cadmium 113 substituted pea and lentil lectins

The lentil (LcH) and pea (PSA) lectins, which are members of the class of D-glucose/D-mannose binding lectins, are Ca2+ X Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. We have prepared a variety of Cd2+ derivatives of PSA and LcH, with Cd2+ in either the transition metal (S1) or calcium (S2) sites, or in both. Thus, Cd2+ X Zn2+, Cd2+ X Mn2+, and Ca2+ X Cd2+ derivatives were prepared, in addition to the Cd2+ X Cd2+ derivatives which we have recently reported. This is the first report of stable mixed metal Cd2+ complexes of lectins. The physical and saccharide binding properties of the Cd2+ derivatives of both lectins were characterized by a variety of physiochemical techniques and found to be the same as those of the corresponding native proteins. 113Cd NMR spectra of mono- and disubstituted 113Cd2+ complexes of LcH and PSA were recorded and compared with 113Cd NMR data for concanavalin A (ConA) (Palmer, A.R., Bailey, D.B., Behnke, W.D., Cardin, A.D., Yang, P.P., and Ellis, P.D. (1980) Biochemistry 19, 5063-5070). The data for the PSA and LcH derivatives were found to be very similar, indicating close homology of their metal ion binding sites. 113Cd resonances at 44.6 ppm and -129.4 ppm for 113Cd2+ X 113Cd2+ X LcH, and at 46.6 and -130.4 for the corresponding PSA derivative, are chemical shifts very similar to those observed for 113Cd2+ X 113Cd2+ X ConA. Assignment of the resonances to the transition metal (S1) and calcium (S2) sites were unambiguous since the Ca2+ X 113Cd2+ and 113Cd2+ X Zn2+ derivatives of both lectins showed single resonances characteristic of the S1 and S2 sites, respectively. The results indicate that, unlike ConA, 113Cd2+ binds tightly to PSA and LcH. Binding of monosaccharide to both lectins induce small (2 ppm) upfield shifts in their S2 113Cd resonances, in contrast to the larger shift (8 ppm) observed in ConA. The 113Cd2+ X Mn2+ complexes of PSA and LcH fail to show a 113Cd resonance characteristic of these derivatives, which provides evidence for the close proximity of the metal ions in the two proteins. The present findings indicate that the coordinating ligand atoms to the metal ions at the S1 and S2 sites in LcH, PSA, and ConA are the same.     

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.     

Behavior of Free Aromatic Amino Acid Pools in Rosmarinic Acid-Producing Cell Cultures of Anchusa officinalis L

The pool sizes of free l-phenylalanine and l-tyrosine, the precursors of rosmarinic acid in Anchusa officinalis L. cell suspension cultures, fluctuated during the culture cycle. The major increase in pool sizes was preceded by a peak of prephenate aminotransferase activity, while the subsequent decrease coincided with the presence of high activities of phenylalanine ammonia-lyase and tyrosine aminotransferase, the two entrypoint enzymes of the rosmarinic acid biosynthesis pathway. Timecourse feeding studies with linear growth stage cells revealed that the tyrosine pool turned over rapidly, consistent with direct participation in rosmarinic acid synthesis. Since externally applied l-tyrosine was rapidly incorporated into rosmarinic acid with little evidence of radioactively labeled intermediates, it is suggested that there exists a close coupling between the l-tyrosine pool and the rosmarinic acid biosynthetic pathway, which may involve the channelling of intermediates both into and within the pathway.     

Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

Effects of cyclopiazonic acid and aflatoxin singly and in combination on selected clinical,pathological and immunological responses of guinea pigs

Cyclopiazonic acid (CPA) and aflatoxin are known sometimes to coexist in nature but little is known of possible biological interaction in mammals that consume mixtures of these two mycotoxins. Guinea pigs were dosed orally with CPA (2.2 mg/kg) or aflatoxin (0.045 mg B₁/kg) singly or in combination. Effects of toxin consumption were determined on clinical health, body weight gain, pathological change, and several immunologically related parameters including delayed cutaneous hypersensitivity, antibody response, complement hemolytic titer, intracutaneous mitogen (PHA) and in vitro lymphocyte blastogenesis. In contrast to an earlier study by others, significant synergy between these two toxins was demonstrated in reduced rate of body weight gain, lethality and histologic changes (vacuolization) in hepatocytes. Reductions in complement titer, intradermal PHA, delayed cutaneous hypersensitivity response and in vitro lymphocyte blastogenesis were related to aflatoxin activity. No effects on antibody formation to Brucella abortus were observed with either toxin or the combination of toxins. Cyclopiazonic acid appeared to restore the suppressive effects of aflatoxin in delayed cutaneous hypersensitivity response and in vitro lymphocyte blastogenesis.     

Some generalized stochastic compartment models for digesta flow

Digesta flow models have been based on linear compartment theory that assumes exponential retention times, and on a generalized theory that incorporates nonexponential (Erlang) retention times (Matis, 1987, Journal of Theoretical Biology 124, 371-376). This paper develops a new family of passage models for heterogeneous digesta by mixing the previous models with assumed parametric, usually gamma, mixing distributions. The utility of the resulting models is demonstrated with experimental data on two treatments, namely a chopped and a ground straw, given to each of four cows. Treatment differences are apparent in the preferred model form and in the means of the estimated mean residence times. The models are relatively easy to fit to data using standard estimation procedures, and they should have broad application to other compartment modeling problems with "heterogeneous particles."     

Activation of pro-urokinase by plasmin: non-Michaelian kinetics indicates a mechanism of negative cooperativity

Enzyme kinetic plots relating the initial rate of activation of pro-urokinase to urokinase by plasmin, according to the concentration of substrate, were smooth downward curves and indicated that an apparent decrease in binding affinity occurred with increase in the concentration of pro-urokinase. Such nonlinear plots were obtained with plasmin 1 and also plasmin 2. Over sections of each curve it was possible to estimate apparent kinetic constants. At the uppermost concentrations of substrate tested, these were Km 2.9 microM and kcat 35.5 min-1 for plasmin 1, and at the lowermost concentrations, Km 9.5 nM and kcat 2.0 min-1. Linear plots were obtained when the single proteolytic cleavage was made by K5-plasmin or undegraded plasmin in the presence of 1.0 mM 6-aminohexanoic acid (6-AHa). Constants were estimated for catalysis of this reaction by K5 plasmin to be Km 6.0 microM and kcat 38 min-1 (r = 0.987). The catalytic efficiency of plasmin, at the lowermost concentrations of pro-urokinase tested, was therefore 33-fold higher than that of K5-plasmin. Plotting of data for the cleavage of pro-urokinase by plasmin 1 (in the absence of 6-AHa) according to the model of Hill, gave a slope of 0.5 at the lowermost concentrations of pro-urokinase increasing to 1.0 at higher concentrations (greater than 0.3 microM); such a profile is characteristic of negative cooperativity. The rates of formation of plasmin and urokinase in a mixture containing a low concentration of plasminogen and pro-urokinase were measured and compared to those predicted by a computer program designed to calculate theoretical rates using available kinetic data. The observed rates of generation of both plasmin and urokinase coincided to those predicted from the negative cooperativity model. The mechanism of the negative cooperativity may reside in a conformational change induced by binding of pro-urokinase to the kringle structure of plasmin. This property may be of significance in controlling the fibrinolytic properties of the urokinase-type plasminogen activator system.     

N2(C2H2−C2H4) fixation in two species of Ceanothus seedlings in second year postfire chaparral

Nitrogen fixation in excised root nodules of 2-year-old, postfireCeanothus tomentosus andC. leucodermis seedlings was measured over an 8-month period using the acetylene reduction method. High levels of NO₃–N and NH₄–N present in postfire soils were limited to the upper 10 cm and did not inhibit nodulation in these deeper-rooting seedlings. Decreases in acetylene reduction activity occurred with decreased soil moisture and increased soil temperature. Nitrogen gains from these two Ceanothus shrub seedlings totalled 1.6 kg N ha^–1 yr^–1.     

Computer-designed prostheses for orbitocranial reconstruction

Three-dimensional imaging is an adjunct to preoperative evaluation and surgical management in some patients with complex anatomic defects of various etiologies. Deformities defined by conventional computerized tomography can be viewed as accurate three-dimensional images calculated from the original scan. The images are viewed on a high-resolution video monitor and can be photographed for a permanent record. A computer-controlled milling device can use these data to fabricate prostheses. The prostheses aid reconstructive surgery through use as an alloplastic implant, as a template to fashion autogenous bone grafts, or as a model for tissue removal. We have utilized three-dimensional imaging in combination with computer-assisted prosthesis manufacture in six patients with complex orbitocranial deformities. Four patients have undergone reconstructive surgery with satisfactory results and no complications thus far. The use of computer-designed prostheses adds a new aspect to orbitocranial reconstructive surgery that facilitates increased accuracy in the correction of anatomic defects.