Gibberellic acid and the light inhibition of stem elongation

Summary The ability of gibberellic acid (GA₃) to prevent the light inhibition of stem elongation in peas was examined for several varieties under a wide range of irradiation conditions.A saturating dose of GA₃ largely prevented the inhibitory effect of red light on total stem height in Duke of Albany (tall), Alaska (medium) and Meteor (dwarf) although a small, but statistically significant, effect persisted in all varieties after 3 days of light. The growth of the second internode was, however, markedly inhibited by red light even with a saturating dose of GA₃. With gibberellin there was no difference between the effects of continuous red light and 15 minutes per day on height but the second internode was much shorter in the former treatment. The number of internodes present was the same in both cases and, therefore, the upper internodes in continuous light were as long or longer than in the 15-minute treatment. The number of internodes was only slightly fewer in darkness than in light so that, with GA₃, the effect of red light was transient and only the growth of the lower internodes was inhibited. Without GA₃ overall height was less in both red light treatments than in darkness for all three varieties.In blue light, on the other hand, there was no difference depending on whether height or internode length is considered, and even with a saturating dose of GA₃ the growth rate remained depressed in continuous blue light. There was, however, some interaction between blue light and GA₃.Red/far-red reversal experiments showed that in the varieties Alska and Duke of Albany the far-red stimulation of elongation persisted in the presence of a saturating dose of GA₃ while for the dwarf variety Meteor there was a significant interaction between far-red and GA₃.At least a quantitative difference was found between tall and dwarf peas in their response to light. Tall varieties showed a much greater effect of a prolonged exposure to blue and a smaller effect of a short exposure to red than dwarf varieties. Increasing the duration of exposure to red increasingly inhibited the growth of tall varieties. The medium variety Alaska grew to approximately the same height in continuous red and blue light.     

Pouch Method for the Isolation and Enumeration of Clostridia

An anaerobic film-pouch method has been developed for the isolation and enumeration of clostridia. Fabrication of the pouch is described. Counts of spore suspensions of Putrefactive Anaerobe 3679 and of Clostridium botulinum strains 41-B and 33-A in pouches were compared with those obtained by anaerobic-jar and agar-deep techniques. Statistical analysis revealed a significant difference in favor of the pouch over the tube and anaerobic-jar methods. Tests performed with C. welchii, both in spore suspension and added to chicken pot pie in culture form, also demonstrated the pouch to be at least as proficient as the other, more cumbersome, techniques.     

Incidence of Mesophilic Clostrodium Spores in Raw Pork, Beef, and Chicken in Processing Plants in the United States and Canada

The anaerobic film pouch technique was used to quantitate and isolate clostridial spores in 2,358 samples of raw meat (1,078 of chicken, 624 of beef, 656 of pork). Of 19,727 putrefactive anaerobic (PA) sporeformers isolated, 1 was confirmed by mouse protection testing to be Clostridium botulinum type C. This isolate was obtained from a Western Canada chicken sample which contained 5.33 clostridia per gram. These data indicate a very low incidence of botulinal contamination in raw meats at the packing-plant level (0.042% of 2,358 samples) and an almost 20,000:1 ratio of nonbotulinal PA sporeformers to mesophilic C. botulinum spores. The mean level of PA contamination was 2.8 PA sporeformers per gram of meat; 77% of the samples contained three or less PA sporeformers per gram. Small but statistically significant differences in the incidence of clostridial spores were noted for season, geographical region, and type of meat.     

Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus.

Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypically distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], less than 20). A reassortant (E19) that contained EDIM VP7 and WC3 VP4 was also very poorly neutralized by these antisera (GMT = 20). In contrast, antibody titers to a reassortant (R20) that contained WC3 VP7 and rhesus rotavirus VP4 were higher than those against WC3 (GMTs of 458 and 313, respectively). Thus, VP7 appeared to be the dominant immunogen for production of neutralizing antibody after intestinal infection of previously uninfected infants vaccinated with WC3 bovine rotavirus.     

Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge.

A baculovirus-expressed VP4 protein derived from the simian rhesus rotavirus (RRV) was used to parenterally immunize murine dams. VP4-immunized dams developed high levels of neutralizing antibodies against RRV and low levels of cross-reactive neutralizing antibodies against human strains Wa, ST3, and S2 and animal strains SA-11, NCDV, and Eb. Newborn mice suckled on VP4-immunized dams were protected against a virulent challenge dose of the simian strain RRV and against murine rotavirus Eb. The cross-reactive nature of the serum-neutralizing response generated by VP4 immunization and the protective efficacy of the immunization suggest that recombinant-expressed VP4 proteins should be considered as viable vaccine candidates.     

NS35 and not vp7 is the soluble rotavirus protein which binds to target cells.

Recent studies using radiolabeled rotavirus lysates have demonstrated a 35-kilodalton viral protein that binds specifically to the surface of MA104 cells (N. Fukuhara, O. Yoshie, S. Kitakoa, and T. Konno, J. Virol. 62:2209-2218, 1988; M. Sabara, J. Gilchrist, G.R. Hudson, and L.A. Babiuk, J. Virol. 53:58-66, 1985). The binding protein was identified as vp7, an outer capsid glycoprotein and the product of rotavirus gene 9. These studies concluded that vp7 mediated viral attachment to MA104 cells and that the binding of a soluble viral protein to a cell monolayer mirrored the attachment of infectious rotavirus to permissive tissue culture cells. In the process of determining which viral protein adheres to the in vivo target cell in rotavirus infection, the mammalian enterocyte, we found that a similar 35-kilodalton rhesus rotavirus (RRV) protein bound to both MA104 cells and murine enterocytes. However, further analysis of this protein by immunoprecipitation, inhibition of glycosylation, and partial proteolysis showed that it was not the RRV gene 9 product, vp7, but the gene 8 product, NS35. Similar results were obtained by using porcine rotavirus (OSU) and bovine rotavirus (NCDV) strains. Binding studies using the in vitro-expressed products of RRV genes 8 and 9 confirmed these results. Since double-shelled virions inhibited the binding of NS35 to cells, we looked for the presence of this protein in preparations of purified virus. Examination of density gradient-purified virus preparations revealed biochemical and immunological evidence that NS35 copurifies in small amounts with double-shelled virions. Thus, these studies clearly demonstrated that when rotavirus proteins are prepared in a soluble form from infected cells, NS35, and not vp7, binds to the surfaces of MA104 cells and murine enterocytes. The observations do not confirm previous experimental results which supported the hypothesis that vp7 was the viral attachment protein. They are consistent with but do not prove the hypothesis that NS35 functions as the rotavirus attachment protein.     

Sensitivity of lod scores to changes in diagnostic status.

This paper investigates effects on lod scores when one individual in a data set changes diagnostic or recombinant status. First we examine the situation in which a single offspring in a nuclear family changes status. The nuclear-family situation, in addition to being of interest in its own right, also has general theoretical importance, since nuclear families are "transparent"; that is, one can track genetic events more precisely in nuclear families than in complex pedigrees. We demonstrate that in nuclear families log10 [(1-theta)/theta] gives an upper limit on the impact that a single offspring's change in status can have on the lod score at that recombination fraction (theta). These limits hold for a fully penetrant dominant condition and fully informative marker, in either phase-known or phase-unknown matings. Moreover, log10 [(1-theta)/theta] (where theta denotes the value of theta at which Zmax occurs) gives an upper limit on the impact of a single offspring's status change on the maximum lod score (Zmax). In extended pedigrees, in contrast to nuclear families, no comparable limit can be set on the impact of a single individual on the lod score. Complex pedigrees are subject to both stabilizing and destabilizing influences, and these are described. Finally, we describe a "sensitivity analysis," in which, after all linkage analysis is completed, every informative individual in the data set is changed, one at a time, to see the effect which each separate change has on the lod scores. The procedure includes identifying "critical individuals," i.e., those who would have the greatest impact on the lod scores, should their diagnostic status in fact change. To illustrate use of the sensitivity analysis, we apply it to the large bipolar pedigree reported by Egeland et al. and Kelsoe et al. We show that the changes in lod scores observed there, on the order of 1.1-1.2 per person, are not unusual. We recommend that investigators include a sensitivity analysis as a standard part of reporting the results of a linkage analysis.