Application of enzyme flow microcalorimetry to the study of microkinetic properties of immobilized biocatalyst

Summary Flow microcalorimeter was used for the study of microkinetic properties of Escherichia coli cells enriched with the penicillin G acylase activity immobilized in calcium pectate gel. The experimental kinetic data were obtained by measurement of the thermometric signal in the microcalorimetric column with immobilized enzyme and described by the introduced mathematical model involving the mass transfer and reaction kinetic phenomena.     

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles ofSaccharomyces cerevisiae

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures ofSaccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0°C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits featured envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.     

Drug resistance inYarrowia lipolytica

Six strains ofYarrowia lipolytica tested here were resistant to 10–20 g erythromycin or chloramphenicol per L glycerol-agar medium. Cells tolerating 4 g chloramphenicol per L were very rare and reverted rapidly to the highest resistance. In analogy with Ery^R mutants ofKluyveromyces lactis, our strains did not grow at 36°C but did not lose their viability at that temperature. Two levels of resistance were found with oligomycin and antimycin A,i.e. 10 and 3 mg/L in the former and 10 and 2 mg/L in the latter. The higher resistance levels segregated mitotically and were, therefore, controlled extrachromosomally. The lower resistance levels showed very frequent changes from sensitivity to resistance that prevented the genetic analysis of this resistance. An almost continuous range of tolerance to <5–400 μg mucidin per L was found in populations of the strains analyzed. Newly formed Muc^R cells were established only in the presence of the antibiotic. Pure cultures of Muc^R cells showed an extremely high instability caused by their lower viability and very low growth rate in the absence of mucidin. No loss of resistance to antimycin A was found, although Ant^R cells revealed similar negative selection. Mutability Muc^S»Muc^R and Muc^R»Muc^S was higher in Ant^R cells than in Ant^S ones.     

Apoptosis and nutrition: Involvement of amino acid transport system in repression of hybridoma cell death

Mouse hybridoma cells cultured on the verge of starvation-induced apoptosis, i.e. in a medium diluted with saline, proved to serve as a sensitive screening system for apoptosis-suppressing activity of nutrient medium components. Conventional amino acid mixtures were found to suppress the starvation-induced apoptosis, whereas a vitamin mixture was ineffective. (Frank F (1995) Biotechnol. Bioeng. 45: 86–90). Recent experiments showed that suppression of apoptosis, and concurrent resumption of growth, could be achieved by addition of single substances at millimolar concentrations. The set of active substances included certain coded L-amino acids (glycine, alanine, serine, threonine, proline, asparagine, glutamine, histidine), non-coded amino acids (-alanine, taurine, 4-aminobutyric acid), and a non-metabolizable analogue (2-aminoisobutyric acid). This finding shows that some amino acids do not act solely as nutrients, but also as specific signal molecules. The specificity of the effect points to the involvement of adaptively regulated amino acid transport systems A and N in maintaining the balance between triggering and suppression of starvation-induced apoptosis.     

cAMP and adenylate cyclase activity in Streptomyces granaticolor

Abstract Intracellular and extracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels were determined during the growth of Streptomyces granaticolor . The intracellular level of cAMP represents not more than 10% of the total amount. cAMP synthesis varies in cultures growing on different carbon sources. The activity of adenylate cyclase in intact cells is strictly dependent on the presence of a metabolizable carbon source.     

Cytotaxonomic study ofTragopogon L. in czechoslovakia

Karyotypes ofTragopogon orientalis L. subsp.orientalis, T orientalis L. subsp.leiocarpos (Sauter)Trnka,T. pratensis L.,T. minor Miller,T. dubius Scop. subsp.dubius andT. dubius Scop. subsp.major (Jacq.)Vollmann were studied. The occurrence in Slovakia ofT. pratensis was karyologically proved.