Rapid Assay for Bactericidal Antibodies

Microtiter plates were successfully substituted for tubes in the bactericidal test for opsonic antibodies. This method is efficient and should prove useful for screening large numbers of sera.     

Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogue.

The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.     

Apparent in vivo retroconversion of dietary arachidonic to linoleic acid in essential fatty acid-deficient rats

Essential fatty acid-deficient rats were fed ethyl [U-14C]arachidonate (308 dpm/nmol) and when a decrease in the transepidermal water loss was seen, the epidermal sphingolipids, acylglucosylceramide and acylceramide were isolated. [14C]Linoleic acid (approx. 130 dpm/nmol) was present in both lipid classes, while the substrate was only detected in the former. These results intimate that in vivo retroconversion of arachidonic to linoleic acid can be induced in the rat.     

Multiple endo-beta-1,4-glucanase-encoding genes from Bacillus lautus PL236 and characterization of the celB gene

A Bacillus lautus strain was isolated from compost by its ability to degrade microcrystalline Avicel cellulose and acid-swollen cellulose. Three DNA fragments cloned in Escherichia coli encoded at least four endo-beta-1,4-glucanases (EG), of which at least two were contained on one DNA fragment. Another fragment, of 2.5 kb and carrying celB, was cloned in the shuttle-vector plasmid, pJKK3-1, and expressed in E. coli and Bacillus subtilis. The fragment was sequenced and shown to encode a 62-kDa protein, which was found as a 56-kDa mature and active EG in extracts of E. coli and in the supernatant of B. subtilis. The deduced amino acid (aa) sequence has a homology of 37% identical aa on a stretch of 295 aa to EG-E of Clostridium thermocellum. A low level of homology is detected with the Bacillus-type EG.