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Biomedical subjects

E Freese

Publications and source records attributed to E Freese.

At least 73 records · Page 4Linked to original sources
PubMed1978

Cell binding and growth inhibition by hexachlorophene of decanoate and their reversibility.

More than 80% of the hexachlorophene added to a Bacillus subtilis culture binds to the cells. Complete growth inhibition requires 6 x 10(5) molecules bound per cell. In contrast, more than 99% decanoate remains in solution and 3.8 x 10(7) molecules bound per cell are needed to inhibit growth. Centrifugation and resuspension of cells in growth medium removes only decanoate, whereas the addition of 1% bovine serum albumin to the growth medium removes both inhibitors from their binding sites on the cells. The addition of untreated cells to a hexachlorophene-treated culture enables the hexachlorophene molecules to redistribute among all the cells with the result that the inhibited cells can resume growth.

Bacillus subtilis↗
PubMed1977

Comparison of the effects of two lipophilic acids, hexachlorophene and decanoate, on Bacillus subtilis.

The minimal growth-inhibitory amount of either hexachlorophene (HCP) or decanoate stopped growth, respiration, adenosine 5'-triphosphate synthesis, and amino acid transport of Bacillus subtilis in a culture containing amino acids and citrate as carbon sources. The electron transport system was not affected by this dose. Addition of 27.8 mM glucose or 10 mM malate to an inhibited culture did not reverse the binding of HCP or decanoate to the cells, but it allowed resumption of growth, respiration, and adenosine 5'-triphosphate synthesis, as the glucose or malate then supplied the needed carbon. The addition of glucose or malate did not reverse amino acid transport inhibition caused by decanoate, but it did reverse that due to HCP. However, if the dose of HCP was raised in the presence of glucose or malate, only growth and amino acid transport were affected; this indicates that both HCP and decanoate act at their minimal growth inhibitory doses by inhibiting substrate transport. As active transport of amino acids and ketoacids depends on the proton gradient and the membrane potential of the cells, we conclude that the primary effect of these lipophilic acids is the destruction of the proton-motive force.

Adenosine Triphosphate↗
PubMed1977

Media dependence of commitment in Bacillus subtilis.

At some time during sporulation development, cells of Bacillus subtilis develop a commitment to continue sporulation even after addition of or dilution into a fresh nutrient. The extent of commitment was measured by the titer of spores produced at the time at which the original culture sporulated maximally. Since newly formed spores of B. subtilis soon germinate in the replenished medium, the measurement of their titer, especially of heat-resistant spores, gave low values. This problem was avoided by the germination-delaying effect of methyl anthranilate (1 mM) when added together with the fresh nutrients. In a given culture, the titer of committed cells was then independent of the method by which it was measured, i.e., by the phase-bright, octanol-resistant, or heat-resistant spore titer. The time of commitment depended on the type of nutrient added. Commitment occurred earlor casein hydrolysate. The rates at which non-metabolizable amino acid analogues or the 14C from an amino acid mixture were taken up by the cells increased toward the end of growth and later declined. This decline occurred slowly and was only weakly correlated with the commitment time of an analogous amino acid.

Amino Acids↗
PubMed1977

Sodium effect of growth on aspartate and genetic analysis of a Bacillus subtilis mutant with high aspartase activity.

Most strains of Bacillus subtilis, dervied from the 168 (Marburg) strain, grow slowly on aspartate as sole carbon source. We isolated a mutant (aspH) that grows rapidly on aspartate because it produces aspartase constitutively. Thus, aspartase is needed for rapid growth on aspartate, whereas aspartate-alpha-ketoglutarate aminotransferase is not needed, as was demonstrated by a mutant lacking that enzyme activity. By two--and three-factor crosses using PBSl transduction, the aspH mutation was located between the aroD and the lys markers of the genetic map. Although sodium ions do not affect growth on glucose or L-malate, they specifically stimulate growth on aspartate in both the parent and the aspH mutant strains. Enzyme activities of crude aspartase and fumarase and of purified aspartase do not increase in the presence of sodium. These results show that stimulation by sodium involves some reaction other than the enzymes catabolizing aspartate. The ease of purification from the aspH strain and the stability of aspartase suggest that the B. subtilis enzyme is particularly useful for aspartate determinations.

Ammonia-Lyases↗
PubMed1977

Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis.

Late during sporulation, Bacillus subtilis produces glucose dehydrogenase (GlcDH; EC 1.1.1.47), which can react with D-glucose or 2-deoxy-D-glucose and can use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor. This enzyme is found mainly in the forespore compartment and is present in spores; it is probably made exclusively in the forespore. The properties of GlcDH were determined both in crude cell extracts and after purification. The enzyme is stable at pH 6.5 but labile at pH 8 or higher; the pH optimum of enzyme activity is 8. After inactivation at pH 8, the activity can be recovered in crude extracts, but not in solutions of the purified enzyme, by incubation with 3 M KCl and 5 mM NAD or NADP. As determined by gel filtration, enzymatically active GlcDH has a molecular weight of about 115,000 (if the enzyme is assumed to be globular). GlcDH is distinct from a catabolite-repressible inositol dehydrogenase (EC 1.1.1.18), which can also react with D-glucose, requires specifically NAD as a cofactor, and has an electrophoretic mobility different from that of GlcDH.

Bacillus subtilis↗
PubMed1976

Commitment to sporulation in Bacillus megaterium and uptake of specific compounds.

Commitment of Bacillus megaterium cells to continue the sporulation process was tested at different times during the developmental period with respect to either addition of different carbon sources (sugars or amino acids) or dilution into media containing these. Organisms grown in minimal medium containing sucrose as sole carbon source were committed earliest with respect to aspartic or glutamic acid as sole carbon source, later with respect to fructose, glucose, glycerol or sucrose, and latest with respect to nutrient medium supplemented with casein hydrolysate. Addition of both aspartate and a carbohydrate resulted in later commitment than addition of either compound alone. The initial uptake rates of aspartate, glutamate, glucose and sucrose increased toward the end of growth in complex medium (but not in minimal medium for glucose and sucrose) and then decreased during the developmental period.

Amino Acids↗
PubMed1976

Manganese requirement of phosphoglycerate phosphomutase and its consequences for growth and sporulation of Bacillus subtilis.

In the absence of manganese, rapidly metabolizable carbohydrates such as glucose or glycerol are not completely metabolized by Bacillus subtilis growing in a nutrient sporulation medium: 3-phosphoglyceric acid (3PGA) accumulates inside the cells, growth stops at a low cell titer, and normal sporulation remains suppressed (no prespore septa). Upon the addition of manganese, 3PGA disappears, growth resumes, and normal sporulation takes place. These effects results from a specific manganese requirement of phosphoglycerate phosphomutase which catalyzes the interconversion of 3PGA and 2-phosphoglyceric acid (2PGA). Other metal ions cannot replace manganese, for which the enzyme has an apparent Km of 0.22 mM.

Bacillus subtilis↗
PubMed1975

Morphological changes in cultured mammalian cells: prevention by the calcium ionophore A23187.

The morphological changes induced by butyrate in HeLa cells and by monobutyryl or dibutyryl cAMP in CHO cells are prevented by micromolar concentrations of the divalent cation ionophore A23187. The ionophore is unable to prevent such changes in medium from which calcium is omitted. At slightly higher (but nontoxic) concentrations, the ionophore inhibits the butyrate-mediated induction of the ganglioside biosynthetic enzyme, sialyltransferase, in HeLa. In CHO, sialyltransferase activity is normally high and not altered by any of the compounds tested.

Alkaline Phosphatase↗
PubMed1975

Conditions controlling commitment of differentiation in Bacillus megaterium.

The developmental stage at which cells of Bacillus megaterium are committed to continue differentiation, i.e., sporulation, depends on both the previous growth medium and the new medium to which the cells are transferred for the commitment test. The latest "stage of no return," after which cells continue differentiation, no matter how rich in nutrients the medium, is reached as soon as the forespore is completely surrounded by a double membrane.

Bacillus megaterium↗
PubMed1975

Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells.

Incubation of HeLa cells in the presence of millimolar concentrations of propionate, butyrate, or pentanoate increases the specific activity of CMP-sialic acid:lactosylceramide sialyltransferase 7-20-fold within 24 h. Longer-chain saturated fatty acids or acetate are much less effective, decanoate showing no induction. Unsaturated fatty acid analogs of butyrate and other compounds are ineffective. Only the three most effective compounds also produce characteristic smooth extended cell processes in HeLa cells. Butyrate (5 mM) induces the sialyltransferase after a 4-h lag, producing maximum specific activity by 24 h. The amount of sialyl-lactosylceramide, the glycolipid product of the enzyme, increases during that time 3.5 times more than in control cultures. No other glycosphingolipid enzyme is significantly altered by butyrate exposure. The cellular shape changes occur 2-3 h later than the increase of sialyltransferase activity, and both processes require the continuous presence of inducer and the synthesis of RNA and protein but not the synthesis of DNA or the presence of serum.

Blood Proteins↗
PubMed1975

Inhibitory effects of lipophilic acids and related compounds on bacteria and mammalian cells.

The inhibitory effect of lipophilic acids, antimicrobial food additives, and analgesics-antipyretics was examined at concentrations from 0.1 to 100 mM in bacteria (Bacillus subtilis and Escherichia coli) and mammalian cells (HeLa, human fibroblasts, and mouse neuroblastoma cells). Most compounds inhibit the growth of HeLa cells about as efficiently as that of B. subtilis. However, butyrate and propionate, as well as acetaminophen, antipyrene, phenacetin, and salicylamide, inhibit HeLa at millimolar concentrations whereas, at least 10 times higher concentrations are needed to inhibit B. subtilis. The concentrations needed to inhibit growth by 50% decrease with increasing octanol-water partition coefficients of the compound. Growth of E. coli is inhibited similar to that of B. subtilis by all compounds except butylbenzoate, decanoate, and linoleate which cannot penetrate the lipopolysaccharide layer. All growth inhibitors inhibit amino acid uptake into bacteria and their vesicles, and oxygen consumption in bacteria. In HeLa cells or human fibroblasts, neither amino acid uptake nor adenine 5'-triphosphate synthesis are inhibited by fatty acids at concentrations that completely inhibit growth. Short chain fatty acids (propionate, butyrate, and pentanoate) induce in HeLa the formation of cell processes. In neuroblastoma cells, grown in the presence of 10% fetal calf serum, butyrate also induces such processes which slowly continue to grow in length for at least 7 days; these processes differ in speed of formation, width, and cycloheximide susceptibility from the thin processes produced by serum deprivation alone.

Bacteria↗
PubMed1975

Motility of Bacillus subtilis during growth and sporulation.

The change of motility and the presence of flagella were followed throughout growth and sporulation in a standard sporulating strain and in 19 cacogenic sporulation mutants of Bacillus subtilis. For the standard strain, the fraction of motile cells decreased during the developmental period to less than 10% at T4. Motility was lost well before the cells lose their flagella. Conditions reducing the decrease of motility also reduced sporulation: motile cells never contained spores. The decrease of motility was not coupled with a decrease in the cellular concentration of adenosine 5'-triphosphate or a decline in oxygen consumption, but an uncoupling agent immediately destroyed motility at any time. Apparently, motility decreased during development because it became increasingly uncoupled from the energy generating systems of the cell. The motility of sporulation mutants decreased after the end of growth at the same time as or earlier than the motility of the standard strain; the early decrease of motility in an aconitase mutant, but not that in an alpha-ketoglurate dehydrogenase mutant, could be avoided by addition of L-glutamate. Sporulation or related events such as extracellular antibiotic or protease production were not needed for the motility decline.

Adenosine Triphosphate↗
PubMed1974

Deficiencies or excesses of metabolites interfering with differentiation.

Auxotrophic mutants of Bacillus subtilis need much higher concentrations of the required adenine, nicotinic acid, riboflavin, thiamine, or tryptophan for optimal sporulation than for maximal growth. Acetate can partially replace thiamine, indicating the importance of the pyruvate dehydrogenase system for differentiation. A glycerol-requiring mutant can sporulate only if its cells contain a small concentration of L-alpha-glycerol phosphate during development. This can best be achieved by excess (>/=5 mM) of extracellular alpha-glycerol phosphate, which enters B. subtilis very slowly. The results show that both biosynthetic and catabolic enzymes are often needed to maintain the precise balance of metabolites required for differentiation. Mutants unable to catabolize fructose 6-phosphate, glucose 6-phosphate, or alpha-glycerol phosphate do not sporulate as long as these compounds accumulate inside the cells; their development is blocked before prespore septa have formed.

Bacillus subtilis↗
PubMed1974

Explanation for the apparent inefficiency of reduced nicotinamide adenine dinucleotide in energizing amino acid transport in membrane vesicles.

Lineweaver-Burk plots of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane preparations from Bacillus subtilis are biphasic, with two K(m) values for NADH. The higher K(m) corresponds to the only K(m) observed for NADH oxidation by whole cells, whereas the lower K(m) corresponds to that observed with open cell envelopes. Membrane preparations apparently contain a small fraction of open or inverted vesicles which is responsible for the low K(m) reaction, whereas entry of NADH into the larger portion of closed, normally oriented vesicles is rate limiting and responsible for the high K(m) reaction. In contrast, the oxidation of l-alpha-glycerol-phosphate (glycerol-P) by membrane preparations shows only one K(m) that corresponds to that of glycerol-P oxidation by whole cells or lysates. Since glycerol-P dehydrogenase (NAD independent) has the same K(m), this enzyme reaction rather than entry of glycerol-P into vesicles represents the rate-limiting step for glycerol-phosphate oxidation. The K(m) for amino acid uptake by vesicles in the presence of NADH corresponds to the high K(m) for NADH oxidation, indicating that NADH energizes transport only if it enters closed, normally oriented vesicles. Studies with rotenone and proteolytic enzymes support this interpretation. The apparent efficiency of NADH in energizing uptake seems to be lower than that of glycerol-P because, under the experimental conditions usually employed, open or inverted vesicles that do not participate in amino acid uptake are responsible for the major portion of NADH oxidation. When the results are corrected for this effect, the efficiency of NADH is essentially the same as that of l-alpha-glycerol-P.

Amino Acids↗