Observations on enzymes of ammonia assimilation in two different strains of Cyanidium caldarium

Two strains of Cyanidium caldarium, one able to utilize nitrate as a substrate, and the other not, were tested for the presence of enzymes of ammonia assimilation. The nitrate-assimilating strain exhibits glutamate dehydrogenase activity. By contrast, the other strain lacks glutamate dehydrogenase; it possesses high alanine dehydrogenase and l-alanine aminotransferase activities which suggest that this strain may incorporate ammonia through reductive amination of pyruvate and may form glutamate from 2-ketoglutarate by a transamination reaction with alanine. Neither strain reveals glutamate synthase activity. Both strains contain similar levels of glutamine synthetase.     

Effect of nitrate,ammonia and nitrogen starvation on the regulation of nitrate reductase in Cyanidium caldarium

Summary Cells of Cyanidium caldarium grown with ammonia or ammonium nitrate as nitrogen source do not contain appreciable nitrate reductase activity. The alga develops the capacity to synthesize the enzyme when it is transferred from the ammonium medium to a nitrogen-free medium. Nitrate is not needed as an inducer and no enhancement in the rate of enzyme synthesis is observed when it is present. By contrast, whereas the synthesis of the enzyme in nitrogen-free medium proceeds at an increasing rate, in the nitrate medium it attains a stationary level after a short time.Nitrate grown cells possess variable amount of inactive nitrate reductase (from 9 to 60%) whereas in nitrogen-free medium the enzyme occurs principally in a fully active form. Addition of ammonia inactivates reversibly the preexisting enzyme. The inactive enzyme is measurable in the crude extract after activation by heating.It is suggested that in Cyanidium the inactivating effect of ammonia, which is the end product of nitrate reduction, in association with the repression of enzyme controls the level of nitrate reductase activity.     

Polymorphonuclear leukocyte migration through human amnion membrane

A new in vitro model has been developed for studying migration of human polymorphonuclear leukocytes (PMN) through living native cellular and matrix barriers. Human amnion membrane consists of a single layer of epithelium bound to a continuous basement membrane interfacing an avascular collagenous stroma. Living amnion was placed in plastic chambers with separate compartments on each side of the membrane. PMN were introduced on the epithelial side of the amnion, and a Millipore filter (Millipore Corp., Bedford, Mass.) was placed against the stromal side. In response to N-formylmethionyl-leucyl- phenylanlanine (FMLP) chemoattractant, PMN penetrated the full thickness of the amnion and were collected and counted on the filter. The rate of PMN traversal of the amnion was dependent on the concentration of FMLP (optimal at 10(-8)M) as well as the slope of the FMLP gradient across the amnion. The route of PMN migration was studied by transmission electron microscopy. PMN first attached to the epithelial surface, then infiltrated between intercellular junctions. PMN migrated around or through tight junction and hemidesmosome attachments. The PMN then penetrated the basement membrane and migrated through the dense collagenous stroma. The present amnion migration system has characteristics of the in vivo inflammatory state not described in any previous method for monitoring PMN migration in vitro. Prior methods have not used native epithelium, whole basement membrane, or collagenous stroma. PMN penetration of these barriers occurs in the normal inflammatory response and probably involves biochemical mechanisms not required for simple migration through the pores of an artificial filter. The amnion system can be useful for future biochemical and morphological studies of PMN penetration of these barriers and possible repair processes that may follow.