SUMMARY:A sensitive spectrophotometric test has been developed to rapidly quantitate cell growth and viability on monolayer cultures. The method consists of staining fixed cells with the supravital dye Janus green, extraction of the dye from these cells with absolute alcohol, spectrophotometric measurement of the eluant, and referring optical density (OD) readings to a previously established standard curve such that measurements can be expressed directly as number of surviving cells. Staining of cells with the same colorant before fixation allows microscopical or colorimetric quantitation or both of damaged cells still adherent to the substratum. Both steps can be conveniently carried out on the same cell culture plate. More rapid and easier to perform than the usual cell counting or tritiated thymidine assays, this technique may prove to be useful in evaluation of the effect of growth promoting substances, growth inhibitors, or cytotoxic agents on monolayer cultures.

New information about the proteins of the phosphotransferase system (PTS) and of phosphoglycosidases of homofermentative lactic acid bacteria and related species is presented. Tertiary structures were elucidated from soluble PTS components. They help to understand regulatory processes and PTS function in lactic acid bacteria. A tertiary structure of a membrane-bound enzyme II is still not available, but expression of Gram-positive genes encoding enzymes II can be achieved in Escherichia coli and enables the development of effective isolation procedures which are necessary for crystallization experiments. Considerable progress was made in analysing the functions of structural genes which are in close vicinity of the genes encoding the sugar-specific PTS components, such as the genes encoding the tagatose-6-P pathway and the 6-phospho-beta-glycosidases. These phosphoglycosidases belong to a subfamily of the beta-glycosidase family I among about 300 different glycosidases. The active site nucleophile was recently identified to be Glu 358 in Agrobacterium beta-glucosidase. This corresponds to Glu 375 in staphylococcal and lactococcal 6-phospho-beta-galactosidase. This enzyme is inactivated by mutating Glu 375 to Gln. Diffracting crystals of the lactococcal 6-P-beta-galactosidase allow the elucidation of its tertiary structure which helps to derive the structures for the entire glycosidase family 1. In addition, a fusion protein with 6-phospho-beta-galactosidase and staphylococcal protein A was constructed.