Colorimetric enumeration of Escherichia coli based on beta-glucuronidase activity

A medium containing a chromogenic substrate was developed for the enumeration of Escherichia coli on the basis of beta-glucuronidase activity. In this medium there was an inverse linear relationship between the log initial E. coli concentration and the time taken for the color to reach a threshold optical density of 0.05. This relationship applied even when the E. coli population contained 5% beta-glucuronidase-negative cells. Incubation at 44 degrees C reduced the time taken for color development and allowed the procedure to be used in the presence of a competitive microflora that outnumbered the E. coli population by a factor of 10(4). Sodium lauryl sulfate as an additional selective agent gave no significant improvement. In the analysis of environmental water samples, the technique gave a good correlation with a standard cultural method. The procedure shows promise as a simple method for testing the compliance of environmental samples with microbiological criteria for E. coli.     

The ontogeny of immunoreactive beta-endorphin and beta-lipotropin in the rat testis

We have investigated the ontogeny of immunoreactive beta-endorphin (i-beta E) in the testes of rats from 5 to 150 days of age. i-beta E was measured by RIA in acid extracts of decapsulated testes and characterized by gel filtration chromatography. Significant age-related differences in both the levels and type of i-beta E were observed. Total levels of i-beta E in the testes were very low and barely detectable from 5-20 days of age, but rose sharply in parallel with testes weights from 20-60 days of age; thereafter, no significant changes in i-beta E were found through 150 days of age. Concentrations of i-beta E, expressed in pmol/g testis, fell precipitously between days 5 and 10 and remained relatively constant from 10-150 days. Most of the i-beta E at 5 and 15 days chromatographed like authentic beta-endorphin. However, with the onset of puberty (30-35 days) and during sexual maturation, much of the total i-beta E chromatographed like its' precursor beta-lipotropin (beta LPH). Hypophysectomy decreased the weight and total i-beta E levels of the testes to the same extent without altering the concentrations of i-beta E or the chromatographic pattern of i-beta E. These results indicate that beta E-like and beta LPH-like peptides are present in the rat testis and that age-related changes in both the levels and type of i-beta E correlate with various structural and functional aspects of testicular development.     

Characterization of an extremely thermostable glutamate dehydrogenase: a key enzyme in the primary metabolism of the hyperthermophilic archaebacterium, Pyrococcus furiosus.

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3) from the hyperthermophilic Archeon Pyrococcus furiosus was purified to homogeneity by chromatography on anion-exchange, molecular-exclusion and hydrophobic-interaction media. The purified native enzyme had an M(r) of 270,000 +/- 15,000 and was shown to be a hexamer with identical subunits of M(r) 46,000. The enzyme was exceptionally thermostable, having a half-life of 3.5 to more than 10 h at 100 degrees C, depending on the concentration of enzyme. The Km of the enzyme for ammonia was high (9.5 mM), indicating that the enzyme is probably active in the deaminating, catabolic direction. The coenzyme utilization of the enzyme resembled the equivalent enzymes from eukaryotes rather than eubacteria, since both NADH and NADPH were recognized with high affinity. The enzyme displayed a preference for NADP+ over NAD+ that was more pronounced at low assay temperatures (50-70 degrees C) compared with the optimal temperature for enzyme activity, 95 degrees C.     

The carotenoid zeaxanthin and ‘high-energy-state quenching’ of chlorophyll fluorescence

The possibility that zeaxanthin mediates the dissipation of an excess of excitation energy in the antenna chlorophyll of the photochemical apparatus has been tested through the use of an inhibitor of violaxanthin de-epoxidation, dithiothreitol (DTT), as well as through the comparison of two closely related organisms (green and blue-green algal lichens), one of which (blue-green algal lichen) naturally lacks the xanthophyll cycle. In spinach leaves, DTT inhibited a major component of the rapidly relaxing high-energy-state quenching' of chlorophyll fluorescence, which was associated with a quenching of the level of initial fluorescence (F₀) and exhibited a close correlation with the zeaxanthin content of leaves when fluorescence quenching was expressed as the rate constant for radiationless energy dissipation in the antenna chlorophyll. Green algal lichens, which possess the xanthophyll cycle, exhibited the same type of fluorescence quenching as that observed in leaves. Two groups of blue-green algal lichens were used for a comparison with these green algal lichens. A group of zeaxanthin-free blue-green algal lichens did not exhibit the type of chlorophyll fluorescence quenching indicative of energy dissipation in the pigment bed. In contrast, a group of blue-green algal lichens which had formed zeaxanthin slowly through reactions other than the xanthophyll cycle, did show a very similar response to that of leaves and green algal lichens. Fluorescence quenching indicative of radiationless energy dissipation in the antenna chlorophyll was the predominant component of high-energy-state quenching in spinach leaves under conditions allowing for high rates of steady-state photosynthesis. A second, but distinctly different type of high-energy-state quenching of chlorophyll fluorescence, which was not inhibited by DTT (i.e., it was zeaxanthin independent) and which is possibly associated with the photosystem II reaction center, occurred in addition to that associated with zeaxanthin in leaves under a range of conditions which were less favorable for linear photosynthetic electron flow. In intact chloroplasts isolated from (zeaxanthin-free) spinach leaves a combination of these two types of rapidly reversible fluorescence quenching occurred under all conditions examined.Abbreviations DTT dithiothreitol - F₀ (or F₀) yield of instantaneous fluorescence at open PS II reaction centers in the dark (or during actinic illumination) - F_M (or F_M) yield of maximum fluorescence induced by a saturation pulse of light in the dark (or during actinic illumination) - F_V (or F_V) yield of variable fluorescence induced by a saturating pulse of light in the dark (or during actinic illumination) - k _D rate constant for radiationless energy dissipation in the antenna chlorophyll - SV Stern-Volmer equation - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q_A acceptor of photosystem II - q_N coefficient of nonphotochemical chlorophyll fluorescence quenching - q_P coefficient of photochemical chlorophyll fluorescence quenching     

Ventilatory control during exercise in calves with artificial hearts

To determine the role of cardiac reflexes in mediating exercise hyperpnea, we investigated ventilatory responses to treadmill exercise in seven calves with artificial hearts and seven controls. In both groups, the ventilatory responses were adequate for the metabolic demands of the exercise; this resulted in regulation of arterial PCO2 and pH despite the absence of cardiac output increase in the implanted group. In this group, there was a small but significant reduction of arterial PO2 by 4 +/- 3 Torr and a rise of blood lactate by 1.1 +/- 1 mmol/l. When cardiac output was experimentally increased in the implanted calves to a level commensurate with that spontaneously occurring in the control calves, ventilation was not affected. However, experimental reductions of cardiac output led to an immediate augmentation of exercise hyperpnea by 4.56 +/- 4.3 l/min and a further significant lactate increase of 1.2 +/- 1.22 mmol/l that was associated with a significant decrease in the exercise O2 consumption (0.32 +/- 0.13 l/min). These observations indicate that neither cardiac nor hemodynamic effects of increased cardiac output constitute an obligatory cause of exercise hyperpnea in the calf.     

Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga maritima.

Previous studies have shown that the hyperthermophilic archaeon Pyrococcus furiosus contains four distinct cytoplasmic 2-ketoacid oxidoreductases (ORs) which differ in their substrate specificities, while the hyperthermophilic bacterium Thermotoga maritima contains only one, pyruvate ferredoxin oxidoreductase (POR). These enzymes catalyze the synthesis of the acyl (or aryl) coenzyme A derivative in a thiamine PPi-dependent oxidative decarboxylation reaction with reduction of ferredoxin. We report here on the molecular analysis of the POR (por) and 2-ketoisovalerate ferredoxin oxidoreductase (vor) genes from P. furiosus and of the POR gene from T. maritima, all of which comprise four different subunits. The operon organization for P. furiosus POR and VOR was porG-vorDAB-porDAB, wherein the gamma subunit is shared by the two enzymes. The operon organization for T. maritima POR was porGDAB. The three enzymes were 46 to 53% identical at the amino acid level. Their delta subunits each contained two ferredoxin-type [4Fe-4S] cluster binding motifs (CXXCXXCXXXCP), while their beta subunits each contained four conserved cysteines in addition to a thiamine PPi-binding domain. Amino-terminal sequence comparisons show that POR, VOR, indolepyruvate OR, and 2-ketoglutarate OR of P. furiosus all belong to a phylogenetically homologous OR family. Moreover, the single-subunit pyruvate ORs from mesophilic and moderately thermophilic bacteria and from an amitochondriate eucaryote each contain four domains which are phylogenetically homologous to the four subunits of the hyperthermophilic ORs (27% sequence identity). Three of these subunits are also homologous to the dimeric POR from a mesophilic archaeon, Halobacterium halobium (21% identity). A model is proposed to account for the observed phenotypes based on genomic rearrangements of four ancestral OR subunits.     

Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length.     

Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene.

Cyclin D1 is the regulatory subunit of certain protein kinases thought to advance the G1 phase of the cell cycle. Deregulated cyclin D1 expression has been implicated in several human neoplasms, most consistently in centrocytic B lymphoma, where the cyclin D1 gene usually has been translocated to an immunoglobulin locus. To determine directly whether constitutive cyclin D1 expression is lymphomagenic, transgenic mice were generated having the cyclin D1 gene linked to an immunoglobulin enhancer. Despite abundant transgene expression, their lymphocytes were normal in cell cycle activity, size and mitogen responsiveness, but young transgenic animals contained fewer mature B- and T-cells. Although spontaneous tumours were infrequent, lymphomagenesis was much more rapid in mice that co-expressed the cyclin D1 transgene and a myc transgene than in mice expressing either transgene alone. Moreover, the spontaneous lymphomas of myc transgenic animals often ectopically expressed the endogenous cyclin D1 gene. These findings indicate that this G1 cyclin can modulate differentiation and collaborate with myc-like genes in oncogenesis.     

Common mutations in the phosphofructokinase-M gene in Ashkenazi Jewish patients with glycogenesis VII--and their population frequency.

Phosphofructokinase (PFK) catalyzes the rate-limiting step of glycolysis. Deficiency of the muscle enzyme is manifested by exercise intolerance and a compensated hemolytic anemia. Case reports of this autosomal recessive disease suggest a predominance in Ashkenazi Jews in the United States. We have explored the genetic basis for this illness in nine affected families and surveyed the normal Ashkenazi population for the mutations we have found. Genomic DNA was amplified using PCR, and denaturing gradient-gel electrophoresis was used to localize exons with possible mutations. The polymorphic exons were sequenced or digested with restriction enzymes. A previously described splicing mutation, delta 5, accounted for 11 (61%) of 18 abnormal alleles in the nine families. A single base deletion leading to a frameshift mutation in exon 22 (delta C-22) was found in six of seven alleles. A third mutation, resulting in a nonconservative amino acid substitution in exon 4, accounted for the remaining allele. Thus, three mutations could account for all illness in this group, and two mutations could account for 17 of 18 alleles. In screening 250 normal Ashkenazi individuals for all three mutations, we found only one delta 5 allele. Clinical data revealed no correlation between the particular mutations and symptoms, but male patients were more symptomatic than females, and only males had frank hemolysis and hyperuricemia. Because PFK deficiency in Ashkenazi Jews is caused by a limited number of mutations, screening genomic DNA from peripheral blood for the described mutations in this population should enable rapid diagnosis without muscle biopsy.