Purification and characterization of ATP:AMP phosphotransferase from Mycobacterium marinum

ATP:AMP phosphotransferase (EC 2.7.4.3) (adenylate kinase) has been purified 1746-fold from Mycobacterium marinum (ATCC 927) by successive column chromatography on DEAE-cellulose (DE-53), Reactive Blue agarose, Sephadex G-75, hydroxyapatite and, finally, DEAE-Sephadex A-50. The final enzyme preparation had a specific activity of 576 mumol/min per mg protein with an overall yield of 51%. The preparation was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was estimated to have an Mr of 29500 and an isoelectric point of 6.7, properties which generally resemble those of the mitochondrial enzyme. Indeed, the two enzymes failed to separate when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The extinction coefficient (at 276 nm) was calculated to be 3.114 X 10(4) M-1 X cm-1 and E1%1cm = 10.556. Adenylate kinase was present at a concentration of 0.06 mg/g (wet weight) bacteria. Enzyme was stable for months in 60% glycerol in the freezer; at 4 degrees C, less than 5% of the activity was lost over a 7 day period.     

Detection of noncontingent feedback in EMG biofeedback

Noncontingent feedback is frequently used as a placebo control procedure in biofeedback research. Researchers, however, have criticized this procedure for lacking credibility because of easy detection. The present study examined detection of false feedback in biofeedback with EMG. Contingent feedback (CF), truly random false feedback (FF), and controlled false feedback (CFF) groups were compared for changes in EMG levels, report of inaccurate feedback, and report of learning muscle activity reduction. The results indicated that FF procedures are easily detected; therefore, differences found between the FF and CF groups may be influenced by extraneous variables. The CFF group did not detect false feedback, but subjects reported some suspicions in later trials. With more trials, CFF may have also been detected. These results indicate a need for more attention to appropriate placebo control procedures in evaluating the parameters and efficacy of biofeedback.     

Interstrain Variation of the Major Internal Structural Component (p30gag) of Two Murine Oncornaviruses: Comparative Immunochemical, Biochemical, and Biophysical Analysis

The major internal structural protein (p30(gag)) of the Moloney leukemia virus and the endogenous Y-1 murine oncornavirus was examined for biochemical and biophysical manifestations of interstrain antigenic variation. Although the two viral proteins share murine group-specific antigenic determinants, the Y-1 virus p30 appeared to have both a lower relative number of such determinants and a decreased affinity at the cross-reactive sites for Moloney virus p30 monospecific antibodies. Further, immunological analysis indicated the presence of unique antigenic sites on the Moloney virus p30 not shared by the analogous Y-1 virus molecule. The two polypeptides copurified and had similar isoelectric points (pH 6.2 to 6.3) and sedimentation coefficients (2.47S). However, equilibrium sedimentation yielded a significant mass difference between the two proteins, 28,300 +/- 600 and 31,000 +/- 300 daltons for the Moloney and Y-1 virus molecules, respectively. Amino acid analysis indicated a concomitant increase in total residues for the Y-1 virus p30, although a number of residues appeared to have been conserved between the two viral proteins. Conformational studies and hydrodynamic calculations demonstrated marked secondary and tertiary structural differences; with the Y-1 virus p30 being an asymmetric prolate ellipsoid containing 27 to 28% alpha-helix and Moloney virus p30 being somewhat more spherical and possessing an alpha-helical content of 50 to 55%. Two-dimensional mapping of (125)I-labeled tryptic peptides of each p30 suggested that considerable sequence heterogeneity is responsible for many of the biophysical, biochemical, and immunochemical differences in these two analogous structural proteins.     

Synthesis and processing of viral glycoproteins in two nonconditional mutants of Rous sarcoma virus.

We have studied the pattern of glycoprotein synthesis in two nonconditional mutants of Rous sarcoma virus. One mutant, SE33, produces no viral particles but synthesizes Pr92env, which is cleaved intracellularly to mature glycoproteins. The second mutant, SE521, encodes a gPr92env which is not cleaved to gp85 or gp37 and therefore produces virions with the phenotype of Bryan RSV(-) or NY8. Neither of these mutants have detectable genomic deletions. The study of these mutants has led to the following conclusions. (i) In the absence of particle production or p15 synthesis, gPr92env can be cleaved to the mature glycoprotein which is found on the cell surface. (ii) Noncleaved gPr92env is not packaged into virions but is found on the cell surface. (iii) gPr92env alone can account for subgroup specific viral interference. (iv) gPr92env is probably transported to the cell surface before additional glycosylation or cleavage to mature virion glycoprotein. The nonprocessed precursor of SE521 appears to be glycosylated normally, and thus far we have been unable to determine the basis for the defect in this mutant.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

Purification of clinical-grade disulfide stabilized antibody fragment variable—Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli

Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion—toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the V_H or V_L. Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, V_L and V_H PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins—MR1-1 and HA22.     

Gene expression and the evolution of phenotypic diversity in social wasps

Background  

Organisms are capable of developing different phenotypes by altering the genes they express. This phenotypic plasticity provides a means for species to respond effectively to environmental conditions. One of the most dramatic examples of phenotypic plasticity occurs in the highly social hymenopteran insects (ants, social bees, and social wasps), where distinct castes and sexes all arise from the same genes. To elucidate how variation in patterns of gene expression affects phenotypic variation, we conducted a study to simultaneously address the influence of developmental stage, sex, and caste on patterns of gene expression in Vespula wasps. Furthermore, we compared the patterns found in this species to those found in other taxa in order to investigate how variation in gene expression leads to phenotypic evolution.     

An increase in surface area is not required for cell division in early sea urchin development

Cell division requires an increase in surface area to volume ratio. During early development, surface area can increase, volume can decrease, or surface topography can be optimized to allow for division. While exocytosis is thought to be essential for division [Mol. Biol. Cell 10 (1999), 2735; Proc. Natl. Acad. Sci. USA 99 (2002), 3633], exocytosis doesn't always yield an increase in surface area [Proc. Natl. Acad. Sci. USA 79 (1982), 6712]. We used multiphoton laser scanning microscopy, fluorescence spectroscopy, and electron microscopy to monitor membrane trafficking, surface area, volume, and surface topography during early sea urchin development. Despite extensive membrane trafficking monitored by FM 1-43 fluorescence, we find that the net surface area of the embryo does not change prior to the eight-cell stage. During this period, embryo volume decreases by 15%, and microvilli disappear from interior facing membrane segments. Thus, the first three cell divisions utilize residual membrane liberated by decreasing cytoplasmic volume, and reducing microvilli density on interior facing membranes. Only after the eight-cell stage was a net increase in FM 1-43 fluorescence from the embryo surface detected. Our data suggest that compensatory endocytosis is downregulated after this developmental stage to yield an increase in surface area for cell division.     

Rab11b resides in a vesicular compartment distinct from Rab11a in parietal cells and other epithelial cells

The Rab11 family of small GTPases is composed of three members, Rab11a, Rab11b, and Rab25. While recent work on Rab11a and Rab25 has yielded some insights into their function, Rab11b has received little attention. Therefore, we sought to examine the distribution of endogenous Rab11b in epithelial cells. In rabbit gastric parietal cells, unlike Rab11a, Rab11b did not colocalize or coisolate with H(+)/K(+)-ATPase. In MDCK cells, endogenous Rab11b localized to an apical pericentrisomal region distinct from Rab11a. The microtubule agents nocodazole and taxol dramatically alter Rab11a's localization in the cell, while effects on Rab11b's distribution were less apparent. These results indicate that in contrast to Rab11a, the Rab11b compartment in the apical region is not as dependent upon microtubules. While Rab11a is known to regulate transferrin trafficking in nonpolarized cells and IgA trafficking in polarized cells, Rab11b exhibited little colocalization with either of these cargoes. Thus, while Rab11a and Rab11b share high sequence homology, they appear to reside within distinct vesicle compartments.