Evaluation of culture methods to identify bovine feces with high concentrations of Escherichia coli O157

Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log10 CFU/g. The preenrichment streak detected >3.0 log10 CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log10 CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments.     

Dual role for Zn2+ in maintaining structural integrity and inducing DNA sequence specificity in a promiscuous endonuclease

We describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.     

R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+

KpnI REase recognizes palindromic sequence, GGTAC↓C, and forms complex in the absence of divalent metal ions, but requires the ions for DNA cleavage. Unlike most other REases, R.KpnI shows promiscuous DNA cleavage in the presence of Mg²⁺. Surprisingly, Ca²⁺ suppresses the Mg²⁺-mediated promiscuous activity and induces high fidelity cleavage. To further analyze these unique features of the enzyme, we have carried out DNA binding and kinetic analysis. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, Ca²⁺-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg²⁺- and Mn²⁺-mediated reactions and was about three times slower with Ca²⁺. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg²⁺-mediated reactions unlike any other Type II REases, accounting for the promiscuous behavior. R.KpnI, thus displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity in its DNA recognition and cleavage properties.     

Sensitivity of Direct Plating for Detection of High Levels of <Emphasis Type="Italic">E. coli</Emphasis> O157:H7 in Bovine Fecal Samples

To assess the sensitivity of direct plating of bovine fecal samples for detection of Escherichia coli O157:H7, calves (n = 28) were orally inoculated with 10⁹ colony-forming units (cfu) per calf of a mixture of three strains of nalidixic acid-resistant E. coli O157:H7, and fecal samples were collected for analysis. One-gram samples from inoculated calves were mixed with 9 mL of Gram-negative broth with vancomycin, cefixime, and cefsoludin. From this suspension, serial dilutions were made (10⁻¹ to 10⁻⁴) and spread plated in triplicate on Sorbitol MacConkey agar with nalidixic acid for enumeration of E. coli O157:H7 in fecal samples. Direct plating samples were streaked for isolation on Sorbitol MacConkey agar with cefixime, and tellurite (SMACct). After incubation overnight at 37°C, morphologically typical colonies from direct streak plates were plated onto blood agar and incubated overnight at 37°C; then an indole test was performed on each colony. Indole-positive colonies were confirmed by O157 agglutination and were then plated on SMAC agar with 20 μg/mL nalidixic acid (SMACnal) to confirm nalidixic acid resistance. Overall sensitivity of detection was 32.5% (110/338 samples). Sensitivity to detect fecal samples shedding at above 5 × 10⁴ cfu/g was 83% (71/86 samples). Based on these data, direct plating of fecal samples might be an effective way to identify cattle that are likely to be shedding E. coli O157 at high levels.     

Effect of GABAergic substances on firing rate and thermal coefficient of hypothalamic neurons in the juvenile chicken

The goal of the study is to investigate the GABAergic action on firing rate (FR) and temperature coefficient (TC) on hypothalamic neurons in the juvenile chicken. Extracellular recordings were obtained from 37 warm-sensitive, 32 cold-sensitive and 56 temperature-insensitive neurons in brain slices to determine the effect of GABA(A)-receptor agonist muscimol, GABA(A)-receptor antagonist bicuculline, GABA(B)-receptor agonist baclofen and GABA(B)-receptor antagonist CGP 35348. Muscimol and baclofen in equimolar concentrations (1 microM) significantly inhibited FR of the neurons, regardless of their type of thermosensitivity. In contrast, bicuculline, as well as CGP 35348 (10 microM) increased FR of the majority of the neurons. The TC of most chick hypothalamic neurons could not be estimated during muscimol application because FR was completely inhibited. GABA(B)-receptor agonist specifically increased TC. This effect was restricted to cold-sensitive neurons, which were determined in a high number. The TC was significantly increased (p<0.05) by baclofen and significantly decreased (p<0.05) by CGP 35348. The effects of muscimol and baclofen on FR and TC were prevented by co-perfusion of the appropriate antagonists bicuculline and CGP 35348. The results suggest that the fundamental mechanisms of GABAergic influence on temperature sensitive and insensitive neurons in the chicken PO/AH are conserved during evolution of amniotes.     

Applying genetic programming to the prediction of alternative mRNA splice variants

Genetic programming (GP) can be used to classify a given gene sequence as either constitutively or alternatively spliced. We describe the principles of GP and apply it to a well-defined data set of alternatively spliced genes. A feature matrix of sequence properties, such as nucleotide composition or exon length, was passed to the GP system "Discipulus." To test its performance we concentrated on cassette exons (SCE) and retained introns (SIR). We analyzed 27,519 constitutively spliced and 9641 cassette exons including their neighboring introns; in addition we analyzed 33,316 constitutively spliced introns compared to 2712 retained introns. We find that the classifier yields highly accurate predictions on the SIR data with a sensitivity of 92.1% and a specificity of 79.2%. Prediction accuracies on the SCE data are lower, 47.3% (sensitivity) and 70.9% (specificity), indicating that alternative splicing of introns can be better captured by sequence properties than that of exons.     

A mysterious pacemaker suture: an uncommon foreign body reaction

Surgical suture material is usually inert and nontoxic and causes minimal inflammation of tissue. However, foreign body reactions to various suture types can lead to granuloma, abscess, or even sinus formation. We report an elderly female who was incidentally detected to have a mass protruding from the incision site which was confirmed histopathologically a chronic granulomatous reaction to non absorbable suture. The foreign body granulomatous reaction to suture material in the setting of pacemaker implantation has not been described in the literature. We also discuss the existing literature on this underrecognised entity.     

Synthesis, DNA binding, and oxidative cleavage studies of Fe(II) and Co(III) complexes containing bioactive ligands

The two complexes containing bioactive ligands of the type and [Fe(L)] (PF(6))(2) (1) (where L = [1-{[2-{[2-hydroxynaphthalen-1-yl)methylidine]amino}phenyl)imino] methyl}naphthalene-2-ol]) and [Co(L(1)L(2))] (PF(6))(3) (2) (where L(1)L(2) = mixed ligand of 2-seleno-4-methylquinoline and 1,10-phenanthroline in the ratio 1:2, respectively) were synthesized and structurally characterized. The DNA binding property of the complexes with calf thymus DNA has been investigated using absorption spectra, viscosity measurements, and thermal denaturation experiments. Intrinsic binding constant K(b) has been estimated at room temperature. The absorption spectral studies indicate that the complexes intercalate between the base pairs of the CT-DNA tightly with intrinsic DNA binding constant of 2.8 × 10(5) M(-1) for (1) and 4.8 × 10(5) M(-1) for (2) in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2, respectively. The oxidative cleavage activity of (1) and (2) were studied by using gel electrophoresis and the results show that complexes have potent nuclease activity.     

Estimation of nuclear DNA content of various bamboo and rattan species

We determined the nuclear DNA content (genome size) of over 35 accessions each of bamboo and rattan species from Southeast Asia. The 2C DNA per nucleus was quantified by flow cytometry. The fluorescence of nuclei isolated from the leaves and stained with propidium iodide was measured. The genome size of the bamboo species examined was between 2.5 and 5.9 pg DNA per 2C nucleus. The genome size of the rattan species examined ranged from 1.8 to 10.5 pg DNA per 2C nucleus. This information will be useful for scientists working in diverse areas of plant biology such as biotechnology, biodiversity, genome analysis, plant breeding, physiology and molecular biology. Such data may be utilized to attempt to correlate the genome size with the ploidy status of bamboo species in cases where ploidy status has been reported.