Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. I. Dihydrofolate reductase, thymidylate synthetase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae.

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent Km values for dihydrofolate in enzymes from the three strains were in the range of 4.8--7.2 muM and for NADPH 6.5--8.0 muM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10--20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthetase and 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) were similar in the three strains studied.     

Pediococcus cerevisiae mutant with altered transport of folates.

A Pediococcus cerevisiae mutant that actively accumulated folate (PteGlu), in contrast to the wild-type, was also found to exhibit changes in the pattern of uptake of 5-methyl-tetrahydrofolate (5-CH3-H4PteGlu) and amethopterin. Most of the 5-CH3-H4PteGlue accumulated through a glucose- and temperature-dependent process, and a concentrative uptake was also found in gluocse-starved cells and in cells incubated at OC. About 75% of the accumulated 5-CH3-H4PteGlu exchanged with amethopterin. In contrast to the wild type, the mutant accumulated both diastereoisomers of 5-CH3-H4PteGlue by glucose-dependent and glucose-independent processes. Amethopterin and PteGlue competitively inhibited the uptake in both processes, with an apparent lower affinity of the carrier for PteGlu than for the analogue. p-Chloromercuribenzoate strongly inhibited the uptake (75%). The p-chloromercuribenzoate-nonsusceptible and temperature-independent uptake was also competed by amethopterin. Metabolic poisons like sodium azide, potassium fluoride, iodoacetate, and 2,4-dimitrophenol inhibited the glucose-dependent process. Uptake, in the absence of glucose, was enhanced by sodium azide and potassium fluoride.     

Mitochondrial DNA and prehistoric settlements: native migrations on the western edge of North America

We analyzed previously reported mtDNA haplogroup frequencies of 577 individuals and hypervariable segment 1 (HVS1) sequences of 265 individuals from Native American tribes in western North America to test hypotheses regarding the settlement of this region. These data were analyzed to determine whether Hokan and Penutian, two hypothesized ancient linguistic stocks, represent biological units as a result of shared ancestry within these respective groups. Although the pattern of mtDNA variation suggests regional continuity and although gene flow between populations has contributed much to the genetic landscape of western North America, some evidence supports the existence of both the Hokan and Penutian phyla. In addition, a comparison between coastal and inland populations along the west coast of North America suggests an ancient coastal migration to the New World. Similarly high levels of haplogroup A among coastal populations in the Northwest and along the California coast as well as shared HVS1 sequences indicate that early migrants to the New World settled along the coast with little gene flow into the interior valleys.     

Population continuity or replacement? A novel computer simulation approach and its application to the numic expansion (Western Great Basin,USA)

In a previous study, Kaestle and Smith [Am J Phys Anthropol 115 (2001) 1-12] supported a recent (A.D. 1000) Numic expansion into the Great Basin region based on a molecular and statistical analysis of mitochondrial DNA (mtDNA) of ancient and modern native inhabitants of the region. Their statistical methodology could not rule out the possibility that observed differences in haplogroup frequencies are instead the result of long-term microevolutionary change within a single population. To distinguish more effectively between a Numic expansion versus population continuity, we employed a novel computer simulation approach that incorporates microevolutionary factors likely to affect human population genetic variation. We test whether the observed differences in haplogroup frequencies between ancient and modern Great Basin groups could have been produced solely via in situ microevolutionary change. Our results indicate that for reasonable demographic conditions, the observed genetic differences between the observed samples are consistent with population continuity if gene flow among prehistoric Great Basin local groups was less than 1% of local group size per generation. Our analysis also supports a recent population expansion if gene flow between neighboring groups exceeded 8% of local group size per generation. The simulations demonstrate that relatively low gene flow levels and random genetic drift can produce the observed degree of genetic differences between population samples. Although this study focuses on the Numic expansion, this simulation approach can be applied to any geographic region for which genetic data have been collected to address similar questions of population relationships over time.     

Brief communication: Conjoined twins at angel mounds? an ancient DNA perspective

Conjoined twins are born when a single fertilized egg partially splits into two fetuses. A hypothetical case of infant conjoined twins from Angel Mounds, a Middle Mississippian site (A.D. 1050-1400) on the Ohio River near Evansville, Indiana, was discovered in 1941. Morphological analysis does not rule out the field interpretation of this double burial as twins. Ancient mitochondrial DNA recovered from both infants demonstrates that they were not maternal relatives, and hence that they cannot have been conjoined twins.     

Dihydrofolate reductase activity in adriamycin and methotrexate sensitive and resistant P388 leukemia cells

P388 murine leukemia cells 18.4-fold more resistant to methotrexate (MTX) than the parent, drug susceptible line, were shown to possess a 1.5-fold higher dihydrofolate reductase (EC1.5.1.3) (DHFR) activity. This is in contrast to a MTX-resistant line, obtained from adriamycin-resistant cells, which is 27.9-fold more resistant to MTX and exhibits a 22.4-fold higher DHFR activity than that of the parent. The susceptibility of the enzyme to inhibition by MTX does not markedly change with the acquired drug resistance of the cell lines studied. Thus MTX-resistant cells obtained from an adriamycin-resistant line acquired resistance due to increased activity of the target enzyme, whereas other mechanisms are responsible for the resistance of cells derived from the adriamycin-sensitive parent.     

Transport of folinate and related compounds in Pediococcus cerevisiae

The properties of folinate and 5-methyltetrahydrofolate (5-CH(3)-H(4)PteGlu) transport mechanism of Pediococcus cerevisiae were studied. The uptake was dependent on temperature, pH (optimum for both compounds at pH 6.0), and glucose. Iodoacetate, potassium fluoride, and sodium azide inhibited the uptake. 5-CH(3)-H(4)-PteGlu was apparently not metabolized but folinate was metabolized. Metabolism of folinate was reduced by preincubation of cells with fluorodeoxyuridine. The transport system for folinate and 5-CH(3)-H(4)PteGlu were specific for the l-isomers. Pteroylglutamate, aminopterin, and amethopterin did not interfere with the uptake. Tetrahydrofolate competed with the uptake of folinate. The transport of folinate and 5-CH(3)-H(4)PteGlu at 37 C conformed to Michaelis-Menten kinetics; apparent K(m) for both compounds was 4.0 x 10(-7)m, and the V(max) for folinate was 1.0 x 10(-10) moles per min per mg (dry weight) and for 5-CH(3)-H(4)PteGlu it was 1.6 x 10(-10) moles per min per mg (dry weight). Both compounds accumulated in the intracellular pool at a concentration about 80- to 140-fold higher than that in the external medium. Folinate inhibited competitively the uptake of 5-CH(3)-H(4)PteGlu with a K(i) of 0.4 x 10(-7)m. Unlike 5-CH(3)-H(4)PteGlu, which accumulated only at 37 C, folinate was also taken up at 0 C by a glucose- and temperature-independent process, which was not affected by the metabolic inhibitors mentioned above. Since at 0 C the intracellular concentration of folinate was also considerably higher than the external, binding of the substrate to some cellular component is assumed. The finding of an efficient transport system for l-5-CH(3)-H(4)PteGlu is of special interest, since this compound has no growth-promoting activity for P. cerevisiae.     

Effect of folinate on thymidine uptake by Pediococcus cerevisiae

Uptake of (3)H-thymidine by resting cells of Pediococcus cerevisiae was found to be energy- and temperature-dependent. The pH optimum was between 6.5 and 8.0, and after 2 min of incubation most of the radioactivity was found in the deoxyribonucleic acid (DNA) fraction. Iodoacetate at a concentration of 10(-2)m caused a 50% inhibition of uptake. Preincubation of resting cells for 10 min with folinate (10(-3)mu mole/ml) diminished the (3)H-thymidine uptake by 75%. In growing cells, the folinate-induced inhibition was still more striking. Deoxyuridine augmented the folinate effect, whereas fluorodeoxyuridine and aminopterin or amethopterin abolished it. Preincubation with folinate did not interfere with the uptake of (3)H-amethopterin, and thus the inhibitor did not compete for uptake sites within the cell. The role of these inhibitors in reversing the folinate effect is discussed. Cells preincubated with folinate showed an increased incorporation of (14)C-uracil into DNA, presumably after prior conversion to thymidylate. We concluded that the folinate effect was due to stimulation of de novo thymidylate synthesis with concomitant inhibition of the uptake of external thymidine.     

Activity of dihydrofolate reductase in the mollicutesAcholeplasma laidlawii andMycoplasma gallisepticum and their susceptibility to antifolates

Mycoplasma gallisepticum andAcholeplasma laidlawii were found to possess dihydrofolate reductases exhibiting similar specific activities and kinetics, with values in the range of those reported for other microorganisms. The apparent Km values for dihydrofolate in enzymes ofM. gallisepticum andA. laidlawii are 7.95±0.13 and 7.50±0.11 M, and for NADPNH 8.46±0.25 and 9.32±0.18 M, respectively.M. gallisepticum is 3300-fold more resistant to methotrexate than isA. laidlawii; concentrations causing 50% inhibition were 200.00 and 0.06 M, respectively. This is in contrast to almost the same sensitivity to that drug exhibited by the dihydrofolate reductases of both microorganisms.M. gallisepticum is also 3600-fold more resistant to trimethoprim than isA. laidlawii, and the concentrations for 50% inhibition of growth were 1800.0 and 0.5 M, respectively. The high resistance was found to be due partially to a 130-fold lower affinity of the target enzyme for this antifolate, but another mechanism, presumably impaired transport, must also be involved. This is the first report of dihydrofolate reductase activity in Mollicutes.     

The 2007–2010 Q fever epidemic in The Netherlands: characteristics of notified acute Q fever patients and the association with dairy goat farming

We describe the Q fever epidemic in the Netherlands with emphasis on the epidemiological characteristics of acute Q fever patients and the association with veterinary factors. Data from 3264 notifications for acute Q fever in the period from 2007 through 2009 were analysed. The patients most affected were men, smokers and persons aged 40–60 years. Pneumonia was the most common clinical presentation (62% in 2007 and 2008). Only 3.2% of the patients were working in the agriculture sector and 0.5% in the meat-processing industry including abattoirs. Dairy goat farms with Coxiella burnetii-induced abortion waves were mainly located in the same area where human cases occurred. Airborne transmission of contaminated dust particles from commercial dairy goat farms in densely populated areas has probably caused this epidemic. In 2010, there was a sharp decline in the number of notified cases following the implementation of control measures on dairy goat and sheep farms such as vaccination, hygiene measures and culling of pregnant animals on infected farms. In combination with a rise in the human population with antibodies against C. burnetii, these have most likely ended the outbreak. Development of chronic Q fever in infected patients remains an important problem for years to come.