Differential growth rates of Candida utilis mother and daughter cells under phased cultivation.

The yeast Candida utilis was continuously synchronized by the phased method of cultivation with the nitrogen source as the growth-limiting nutrient. The doubling time (phasing period) of cells was 6 h. Both cell number and deoxyribonucleic acid synthesis showed a characteristic stepwise increase during the phased growth. The time of bud emergence coincided with the time of initiation of deoxyribonucleic acid synthesis. Size distribution studies combined with microscopic analysis showed that the cells expanded only during the unbudded phase of growth. Usually the cells stopped increasing in size about 30 min before bud emergence, and the arrest of the increase in cell volume coincided with the exhaustion of nitron from the medium. There was no net change in the volume of cells during the bud expansion phase of growth, suggesting that as the bud expanded, the volume of the mother portion of the cell decreased. After division the cells expanded slightly. The postdivision expansion of cells, unlike the growth before bud initiation, occurred in the absence of the growth-limiting nutrient. The newly formed daughter cells were smaller than the mother cells and expanded at a faster rate, so that both types of cells reached maximum size at the same time. Possible reasons for the different rates of expansion of mother and daughter cells are discussed.     

The calcium-dependent phosphatidylinositol-phosphodiesterase of rat brain. Mechanisms of suppression and stimulation.

1. The activity of the soluble, calcium-dependent phosphatidylinositol-specific phosphodiesterase (EC 3.1.4.10) against [32P]phosphatidylinositol has been investigated. 2. KC1 (only at neutral pH), Mg2+, positively-charged proteins such as histone, and phospholipids containing a choline headgroup are all inhibitory to the enzyme. Choline-phospholipids cause a 90% inhibition at an equimolar ratio to phosphatidylinositol. 3. Other phospholipids (phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid) are all potent stimulators of the enzyme: maximum stimulation being observed at a ratio of 1 mol activator/5--10 mol phosphatidylinositol. 4. Unsaturated amphiphiles such as oleic and oleoyl alcohol also stimulate the activity, maximum stimulation being observed at about an equimolar ratio to phosphatidylinositol. Saturated amphiphiles (such as stearic acid and stearoyl alcohol) are less effective. 5. The activation by acidic phospholipids and unsaturated amphiphiles appear to be independent as they are additive and, under certain conditions, synergistic. 6. Both types of stimulator (independently or together) can reverse the inhibition caused by histone or phosphatidylcholine. 7. Possible mechanisms of the suppression of the phosphatidylinositol phosphodiesterase in vivo, of its activation, and of the amplification of phosphatidylinositol breakdown are discussed.     

The kinetics of rat liver and heart mitochondrial beta-hydroxybutyrate dehydrogenase.

The kinetic mechanisms of the beta-hydroxybutyrate dehydrogenase from rat heart and liver mitochondria were investigated. Both enzymes, show an Ordered Bi Bi mechanism and there are no major differences in the kinetic constants. In both cases, the solubilized enzyme, re-activated with phosphatidylcholine, shows kinetic properties very similar to those of the enzyme bound to the mitochondrial membrane.     

Biopterin

An enzyme that catalyzes the conversion of 2-amino-6-(5'-triphosphoribosyl)amino-5- or 6-formamido-6-hydroxypyrimidine, but not of guanosine triphosphate, to quinonoid 6-(D-erythro-1'-2'-3'-trihydroxypropyl)dihydropterin triphosphate and formic acid has been purified to homogeneity from some mammalian brain and liver. The enzyme of a single strand is a basic protein of 9177 daltons consisting of 68 amino acid residues--except the enzyme from rat brain, which has one additional aspartic acid as residue 7. The enzyme possesses three free SH groups and, in its most active form, 1 mol of phosphate per mole of enzyme. Peptides isolated after hydrolysis with trypsin, chymotrypsin, or weak acid were separated by thin-layer chromatography and sequenced manually by Edman degradation. The complete sequence of the molecule was established as follows: (formula: see text)     

Cell kinetics of a chondrosarcoma.

The cell kinetics of the transplantable DC-II mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: G1, 10-5 hr; S, 9-5 hr; G2, 4 hr with an intermitotic time of 23-5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0-5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.     

Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias.

Five glycosphingolipids (GSL), glucosylceramide, lactosylceramide, trihexosylceramide, globoside, and hematoside (GM3) were studied in serum from normal human subjects and patients with dyslipoproteinemia and found to be exclusively associated with the various classes of serum lipoproteins. Based on a unit weight of lipoprotein protein, the total amount of GSL in serum normal subjects was twice as high in very low density lipoprotein (VLDL) (d less than 1.006 g/ml) and low density lipoprotein (LDL) (d 1.019-1.063 g/ml) as in high density lipoproteins HDL2 (d 1.063-1.125 g/ml) or HDL3 (d 1.125-1.21 g/ml). In abetalipoproteinemia the levels of serum GSL were slightly reduced when compared to normal serum and were all found in the only existing lipoprotein, HDL; this contained 2-3 moles of GSL/ mole of lipoprotein as compared to 0.5 GSL/mole in normal HDL. In hypobetalipoproteinemia and Tangier disease, the serum glycosphingolipids were 10 to 30% reduced in concentration compared to the 75% reduction in other lipids, and were again found to be associated only with the serum lipoproteins. The relative proportions of GSL did not vary substantially in the normo- and hypolipidemic subjects studied. Only in patients with homozygous familial hypercholesterolemia was there a significant (3-4-fold) elevation of all of the five GSL species and this elevation of all of the five GSL species and this elevation correlated well with that of the circulating cholesterol and LDL. On a molar basis the LDL of these patients contained the same amount of GSL as normal subjects (5 moles GSL/mole protein). It is concluded that: (1) glycosphingolipids are associated only with the major lipoprotein classes in both normal and dyslipoproteinemic serum; (2) the relative proportions of the five glycosphingolipids are not significantly affected by dyslipoproteinemia; (3) only in severe hypolipoproteinemia do the remaining serum lipoproteins carry a complement of glycosphingolipids greater than normal. Although our results establish that glycosphingolipids are intimately associated with serum lipoproteins, the mode of association or the structural and functional significance of such an association remains undetermined.     

OLIGOSACCHARIDE STORAGE IN BRAINS FROM PATIENTS WITH FUCOSIDOSIS,GM1-GANGLIOSIDOSIS AND GM2-GANGLIOSIDOSIS (SANDHOFF'S DISEASE)1

Abstract— Analysis of whole autopsy brain from a patient with fucosidosis (α-fucosidase deficiency) revealed minor storage of H-antigen glycolipid [Fuc (α, 1→2) Gal-GlcNAc-Gal-Glc-Ceramide] and a slightly abnormal ganglioside composition in the form of a two-fold elevation of G_M1 and the presence of a fucose-containing glycolipid (a minor component) which co-migrated with G_D1a. The major storage materials in fucosidosis brain were an oligosaccharide (Fuc-Gal-GlcNAc-Man[Fuc-Gal-GlcNAc-Man]-ManGlcNAc) and a disaccharide [Fuc(α, 1→6)-GlcNAc] in the approximate ratio of 5:1. Lesser amounts of a related oligosaccharide (Gal-GlcNAc-Man[Gal-GlcNAc-Man]-Man-GlcNAc) were isolated from the brain of patients with G_M1-gangliosidosis (Types I and II) where the major storage material is known to be G_M1-ganglioside (Gal (β, 1→3)GalNAc(β, 1→4) [NeuNAcf(α, 2→3) Gal(β, 1→4)Glc-Ceramide). Similarly, a related oligosaccharide (GlcNAc-Man [GlcNAc-Man]-Man-GlcNAc) was isolated from the brain of a patient with a total deficiency of N-acetyl-β-d -hexosaminidase (Sandhoff variant of G_M2-gangliosidosis) where the major storage products are known to be G_M2-ganglioside (GalNAc (β 1→4) [NeuNAc (α, 2→3)Gal(β, 1→4)Glc-Ceramine) and its asialo derivative. These studies indicate that glycoproteins containing at least 2 mol of l -fucose per oligosaccharide unit are normally catabolized in human brain. Further, it appears that such glycoproteins are initially catabolized by an endo-N-acetylglucosaminidase to release an oligosaccharide which is then degraded by the sequential action of exo-glycosidases.     

A phospholipid-deacylating system of bacteria active in a frozen medium.

A phosphatidylcholine-deacylating system present in a Butyrivibrio species (probably fibrisolvens) shows appreciable activity at low temperatures with a maximum hydrolysis rate at--10 degrees C. 2. The rate at--10 degrees C is higher than at 39 degrees C unless the system at the latter temperature is stimulated by adding oleic acid or sodium dodecyl sulphate. 3. The low-temperature phospholipase activity has an absolute requirement for thiol reagents, e.g. cysteine, dithiothreitol or mercaptoethanol. 4. Ca2+, Mg2+ and Mn2+ stimulate the activity up to 10 mM, but EDTA inhibits; higher concentrations of Ca2+ also inhibit. 5. The enhancement of activity at low temperatures appears not to be associated with a crystalline change in the hydrated phospholipid substrate, but depends on the formation of a solid phase in the incubation medium which brings the substrate and bacterial cells into juxtaposition or causes fusion.