Murine embryonic antigen (SSEA-1) is expressed on human cells and structurally related human blood group antigen I is expressed on mouse embryos

The stage-specific embryonic antigen (SSEA-1), present on embryonal carcinoma cells and on murine preimplantation embryos, is defined by a monoclonal antibody. The antigenic determinant of SSEA-1 is a carbohydrate structurally related to the human blood group antigen I. Since it has been suggested that the I antigen might represent a precursor or SSEA-1, we used antibodies to SSEA-1 and to I to analyze their expression on mouse preimplantation embryos. Both are expressed on mouse embryos; moreover, I is expressed on earlier embryos than SSEA-1. The I antigen is defined by its expression on human erythrocytes; accordingly, we examined expression of I and SSEA-1 on human peripheral blood elements. We find SSEA-1 to be expressed exclusively on human granulocytes while I is found only on erythrocytes. These results suggest that these closely related antigens can be independently expressed. Analysis of the expression of I and SSEA-1 was then extended to a series of mouse and human cell lines; some express both, some express only one, and some express neither of these antigens. The activation of specific glycosyltransferases and/or glycosidases during development and differentiation appears to be the biochemical mechanism regulating expression of these antigens.     

Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus.

The enzyme carboxyphosphonoenolpyruvate mutase catalyses the formation of one of the two C-P bonds in bialaphos, a potent herbicide isolated from Streptomyces hygroscopicus. The gene encoding the enzyme has been cloned from a subgenomic library from S. hygroscopicus by colony hybridisation using an exact nucleotide probe. An open reading frame has been identified that encodes a protein of molecular mass 32700 Da, in good agreement with the subunit molecular mass of the carboxyphosphonoenolpyruvate mutase recently isolated from this source [Hidaka, T., Imai, S., Hara, O., Anzai, H., Murakami, T., Nagaoka, K. & Seto, H. (1990) J. Bacteriol. 172, 3066-3072]. The gene shares significant sequence similarity with that of phosphoenolpyruvate mutase, an enzyme that catalyses the related interconversion of phosphoenolpyruvate and phosphonopyruvate. When the carboxyphosphonoenolpyruvate-mutase gene was subcloned into the vector pET11a, the mutase was expressed as about 20% of the total soluble cellular protein in Escherichia coli. The mutase has been purified to homogeneity in three steps in 40% yield. With malate dehydrogenase/NADH, (hydroxyphosphinyl)pyruvate gives (hydroxyphosphinyl)lactate (kcat 164 s-1 and Km 680 microM) and this spectrophotometric assay for the product of the mutase reaction has been employed in the mechanistic studies. The kinetics for the mutase reaction have been evaluated for the substrate, carboxyphosphonoenolpyruvate, and for the putative reaction intermediate carboxyphosphinopyruvate, both of which have been prepared by chemical synthesis. Carboxyphosphonoenolpyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 0.020 s-1 and a Km of 270 microM, and carboxyphosphinopyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 7.6 x 10(-4) s-1 and a Km of 2.2 microM. Although the exogenously added intermediate is not kinetically competent, these results suggest that the mechanism for the mutase reaction involves an initial rearrangement to the intermediate carboxyphosphinopyruvate, followed by decarboxylation to yield the product (hydroxyphosphinyl)pyruvate.     

Activation of MAP kinases by calcium-dependent and calcium-independent pathways. Stimulation by thapsigargin and epidermal growth factor.

In order to determine the effect of calcium mobilization on mitogen-activated protein (MAP) kinase activation, we have treated human foreskin fibroblasts (HSWP cells) and human epidermal carcinoma (A431) cells with thapsigargin. Intracellular free calcium was monitored by single cell image analysis using fura-2 and correlated with MAP kinase stimulation as assessed by immunoprecipitation, kinase renaturation assays and immunoblotting. Thapsigargin stimulated the 44- and 42-kDa MAP kinase isozymes in both cell types with kinetics that were slightly delayed relative to enzyme stimulated by epidermal growth factor. Removal of external calcium did not significantly affect the activation of the MAP kinases by thapsigargin, indicating that intracellular calcium mobilization is sufficient to stimulate the enzymes. However, treatment of cells with EGTA under conditions which deplete both intra- and extracellular calcium inhibited stimulation by thapsigargin but not epidermal growth factor. Stimulation of the MAP kinases by the calcium ionophore ionomycin paralleled the activation observed with thapsigargin in both calcium-containing and calcium-free conditions. These results indicate that there are at least two independent pathways for stimulation of MAP kinase: one that is dependent on intracellular calcium mobilization, and one that is mediated by the tyrosine kinase epidermal growth factor receptor and is calcium-independent.     

Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.     

Enumeration of Flexibacter canadensis in environmental samples by using a bacteriophage isolated from soil.

The denitrifier Flexibacter canadensis, in the presence of sulfide, can reduce N2O in the presence of concentrations of C2H2 which normally inhibit N2O reduction. Most-probable-number estimates of naturally occurring F. canadensis populations in various soils and sediments were made with a bacteriophage which is active against and specific for a strain of denitrifying F. canadensis (Is-11). Our survey suggests that F. canadensis is common in the natural environment.     

How can a catalytic lesion be offset? The energetics of two pseudorevertant triosephosphate isomerases

The reaction energetics of four triosephosphate isomerase mutants are compared with those of the wild-type enzyme. The two primary mutants, E165D and H95N, contain site-specific alterations of active site residues. In one case the active site base has been altered (E165D), and in the other, an active site electrophile has been removed (H95N), yet the major effect in each case is the relative destabilization of the transition states for the two chemical (enolization) steps that constitute the catalytic reaction. When the genes encoding each of these sluggish mutant isomerases were subjected to random mutagenesis using chemical reagents and a selection for isomerases of increased catalytic potency was performed, pseudorevertant enzymes with dramatic increases in activity were found. Remarkably, the same second-site suppressor locus partially corrects each lesion. The E165D,S96P pseudorevertant is a 20-fold better catalyst than the E165D mutant from which it is derived, and the H95N,S96P pseudorevertant is about 60 times more active than its H95N parent. The S96P substitution thus increases the catalytic activity in each of two different contexts, H95N and E165D. The energetic consequences of the S96P change are suprisingly similar in each pseudorevertant. The H95N,S96P enzyme is more effective than H95N at stabilizing the intermediate enediol(ate) phosphate and its flanking transition states. The E165D,S96P enzyme likewise stabilizes the transition states for enolization better than E165D, and this pseudorevertant also forms a tighter enzyme-dihydroxyacetone phosphate complex than its parent. These data show how, in these two cases, the catalytic potency of sluggish mutant enzymes can be improved by second-site changes. The results thus provide the beginnings of a detailed understanding of the kinetic refinement of enzyme catalysts.     

Lipid-protein interactions in stacked and destacked thylakoid membranes and the influence of phosphorylation and illumination. Spin label ESR studies

The effects of membrane destacking, protein phosphorylation, and continuous illumination have been studied in pea thylakoid membranes using ESR spectroscopy of an incorporated spin-labelled phosphatidylglycerol. This spin-labelled analogue of an endogenous thylakoid lipid has previously been shown to exhibit a selectivity of interaction with thylakoid proteins. Neither destacking, phosphorylation nor illumination was found to change the ESR spectra appreciably, suggesting that for phosphatidylglycerol at least, neither the number of protein-associated membrane lipids nor their pattern of selectivity was altered. The redistribution of the thylakoid protein complexes in the membrane, under these various conditions, therefore takes place with conservation of the properties of the lipid/protein interface.     

Targeted point mutation that creates a unique Eco RI site within the signal codons of the beta-lactamase gene without altering enzyme secretion or processing

A method has been developed for constructing site-specific mutations by using a strongly selectable marker on which to "piggy-back" a desired mutation that may be phenotypically silent. Using this approach, a new unique Eco RI restriction site has been generated at the beginning of the signal codons of the beta-lactamase gene of the plasmid pBR322. The consequential alteration of the second amino acid of the signal from Ser to Arg has no effect on either the transport or the processing of the beta-lactamase.     

Effects of zomepirac and indomethacin on renal blood flow and electrolyte excretion in anesthetized monkeys

Zomepirac sodium is a new inhibitor of prostaglandin cyclooxygenase with an $\text{in vitro}$ potency equivalent to indomethacin. Since inhibitors of prostaglandin synthesis have marked effects on renal hemodynamics, zomepirac may be expected to reduce renal blood flow (RBF) in a manner similar to indomethacin. This study compares the effects of zomepirac and indomethacin on RBF and electrolyte excretion in anesthetized Rhesus monkeys. Each experiment consisted of a control period followed by 3 or 4 drug treatment periods in which increasing doses of zomepirac (0.5 to 20 mg/kg) or indomethacin (0.5 to 10 mg/kg were given. Indomethacin (5 mg/kg) reduced RBF by 22% and the higher dose (10 mg/kg) reduced RBF by an additional 13%. Zomepirac had little effect on RBF in doses as high as 20 mg/kg. At any given dose the mean plasma concentration of zomepirac was equal to or greater than indomethacin. Peak indomethacin concentration was 48 μg/ml after the 10 mg/kg dose while the peak zomepirac, after 20 mg/kg, was 158 μg/ml. Neither drug had a significant effect on either glomerular filtration rate or excretion rate of sodium or potassium. Thus, zomepirac had only minimal effects on RBF while indomethacin decreased RBF of anesthetized monkeys in a manner qualitatively similar to its effect in other species. The minimal renal effects caused by zomepirac relative to indomethacin in this primate may indicate a therapeutic advantage for zomepirac in man.