Intraprotoplasmic feruloylation of arabinoxylans in Festuca arundinacea cell cultures

Graminaceous primary cell walls contain polysaccharides to which are esterified feruloyl residues. Ester biosynthesis is highly specific and the present experiments were performed to ascertain the likely site of feruloylation in living grass cell cultures. Cell cultures of tall fescue grass (Festuca arundinacea Schreber) incorporated exogenous l-[1-³H]arabinose into polymers at a linear rate after a short lag of approx. 1–3 min. Radiolabelled polymers did not start to accumulate in the culture medium until 20–35 min after [³H]arabinose was supplied. However, polymer-bound feruloyl-arabinose residues began to accumulate ³H after a lag of 1–3 min. Assuming that the onset of secretion of radiolabelled polymers into the medium indicates the time before which essentially all the radiolabel was internal to the plasma membrane, the results show that the polysaccharide-bound [³H]arabinose residues must have been feruloylated within the protoplast.Abbreviations AIR alcohol-insoluble residue - BAW butan1-ol/acetic acid/water (12:3:5 by volume) - BEW butan-1-ol/ ethanol/water (20:5:11 by volume) - EPW ethyl acetate/pyridine/ water (8:2:1 by volume) - R_Ara Chromatographic mobility relative to that of l-arabinose We are very grateful to Mr. Gundolf Wende for assistance with the characterisation of the feruloyl esters. K.E.M. is funded by a studentship from the Science and Engineering Research Council in collaboration with Zeneca Agrochemicals.     

Phytophthora infestans in Poland from 1987–1989; Nuclear DNA Content, Mating Type Distribution and Response to Metalaxyl

Isolates of Phytophthora infestans were collected from all potato growing regions of Poland during the blight seasons of 1987—1989. All 1987 isolates were of Al mating type and were sensitive to metalaxyl. In 1988 and 1989, 46.5 % and 55.3 % of the isolates were sensitive to metalaxyl, respectively. The percentage of highly resistant (R) isolates increased from 25.6 % in 1988 to 39.5 % in 1989; however the percentage of intermediately resistant (I) isolates decreased during that period from 27.9 % to 5.3 %. A significant association was observed between the A1 compatibility type and metalaxyl resistance. The A2 mating type first appeared in 1988, and its frequency increased from 4.7 % of the population in 1988 to 47.6 % in 1989. Coincident with this change in mating type frequency, changes in ploidy levels of isolates were observed. Whereas 3 % of the 1988 isolates were diploid, 90 % of the 1989 A2 isolates and 28.6 % of the 1989 Al isolates were diploid. The approximate 1:1 ratio of the two mating types encountered in 1989, and the predominance of diploidy, indicates that the Polish population of P. infestans has the potential to become sexual.     

Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.     

Relative biological effectiveness measurements using murine lethality and survival of intestinal and hematopoietic stem cells after fermilab neutrons compared to JANUS reactor neutrons and 60Co gamma rays

The relative biological effectiveness (RBE) of the 25-MeV (average energy) neutron beam at the Fermi National Accelerator Laboratory was measured using murine bone marrow (LD50/30) and gut (LD50/6) lethality and killing of hematopoietic colony forming units (CFU-S) or intestinal clonogenic cells (ICC). The reference radiation was 60Co gamma rays. The LD50/30 and LD50/6 for mice exposed to the Fermilab neutron beam were 6.6 and 8.7 Gy, respectively, intermediate between those of JANUS neutrons and 60Co gamma rays. The D0 values for CFU-S and ICC were 47 cGy and 1.05 Gy, respectively, also intermediate between the lowest values found for JANUS neutrons and the highest values found after 60Co gamma rays. The split-dose survival ratios for CFU-S at intervals of 1-6 hr between doses were essentially 1.0 for both neutron sources, while the corresponding split-dose survival ratio for 60Co gamma rays was consistantly above 1, reaching a maximum of 1.7 with a 1-hr interval between doses. The 3-hr split-dose survival ratios for ICC were 1.0 for JANUS neutrons, 1.85 for Fermilab neutrons, and 6.5 for 60Co gamma rays. The RBE estimates for LD50/30 were 1.5 and 2.3 for Fermilab and JANUS neutrons, respectively. Based on LD50/6, the RBEs were 1.9 (Fermilab) and 3.0 (JANUS). The RBEs for CFU-S D0 were 1.4 (Fermilab) and 1.9 (JANUS) and for jejunal microcolony D0 1.4 (Fermilab) and 2.8 (JANUS).     

Incorporation of [14C]cinnamate into hydrolase-resistant components of the primary cell wall of spinach

[¹⁴C]Cinnamate was taken up very rapidly by cultured spinach cells and completely incorporated into low-MW conjugates within 20 min. The ¹⁴C-labelled products were similar whether the [¹⁴C]cinnamate was supplied continuously over a period of hours via a peristaltic pump or instantaneously. Radioactivity was slowly recruited from the low-MW pool into aromatic components of the cell-wall fraction. Saponification of the radioactive wall fraction yielded, in addition to radioactive ferulate and p-coumarate, large amounts of ethyl acetate-soluble radioactive material with the properties of oxidatively coupled phenols. The coupled material was associated with the most highly ‘Driselase’-resistant fractions of the cell wall. In contrast, ‘Driselase’ released most of the wall's ferulate and p-coumarate on disaccharide fragments. It is suggested that the oxidatively coupled phenols are formed from simpler phenols by peroxidase and that they cross-link the polysaccharides to which they are attached, making these polysaccharides relatively ‘Driselase’-resistant.