Real‐time PCR detection of Enterococcus faecalis associated with amyloid arthropathy

Aims: To develop a RT‐PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers. Methods and Results: Bacteria were selected from lesions including AA in layers. The primers were designed based on the phosphate ATP binding cassette transporter (pstS) and xanthine phosphoribosyltransferase (xpt) genes and first tested against three isolates with known base pairs at the specific sites. Subsequently, 12 isolates were selected from our collection by one researcher, and RT‐PCR was performed blinded. The sequence type (ST) was then confirmed by multilocus sequence analysis. Two single‐nucleotide polymorphisms in the pstS and xpt genes allowed an unambiguous identification of ST82. As an alternative to DNA extraction, a boiling method for release of DNA from cells was used. Conclusions: The real‐time PCR targeting ST82 enables rapid screening of Ent. faecalis cultured from suspect cases with results available after a few hours, much faster than multilocus sequence typing and pulse field gel electrophoresis. Significance and Impact of the Study: The new method allows a rapid screening of isolates with results available after only few hours. This RT‐PCR method could be a useful tool for molecular epidemiological studies on the spread of arthropathic and amyloidogenic Ent. faecalis within and between birds more efficiently.     

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.     

Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression

The network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells of Saccharomyces cerevisiae. The cells were grown under both high and low glucose concentrations, i.e., either in a chemostat at steady state with a specific growth rate of 0.1 h(-1) or in a batch culture with a specific growth rate of 0.37 h(-1). Experiments were carried out using [1-(13)C]glucose as the limiting substrate, and the resulting summed fractional labelings of intracellular metabolites were measured by gas chromatography coupled to mass spectrometry. The data were used as inputs to a flux estimation routine that involved appropriate mathematical modelling of the central carbon metabolism of S. cerevisiae. The results showed that the analysis is very robust, and it was possible to quantify the fluxes in the central carbon metabolism under both growth conditions. In the batch culture, 16.2 of every 100 molecules of glucose consumed by the cells entered the pentose-phosphate pathway, whereas the same relative flux was 44.2 per 100 molecules in the chemostat. The tricarboxylic acid cycle does not operate as a cycle in batch-growing cells, in contrast to the chemostat condition. Quantitative evidence was also found for threonine aldolase and malic enzyme activities, in accordance with published data. Disruption of the MIG1 gene did not cause changes in the metabolic network structure or in the flux pattern.     

A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.     

The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas

A comprehensive analysis of somatic and germline mutations related to DNA mismatch-repair (MMR) genes can clarify the prevalence and mechanism of inactivation in colorectal carcinoma (CRC). In the present study, 257 unselected patients referred for CRC resection were examined for evidence of defective DNA MMR. In particular, we sought to determine the frequency of hereditary defects in DNA MMR in this cohort of patients. MMR status was assessed by testing of tumors for the presence or absence of hMLH1, hMSH2, and hMSH6 protein expression and for microsatellite instability (MSI). Of the 257 patients, 51 (20%) had evidence of defective MMR, demonstrating high levels of MSI (MSI-H) and an absence of either hMLH1 (n=48) or hMSH2 (n=3). All three patients lacking hMSH2, as well as one patient lacking hMLH1, also demonstrated an absence of hMSH6. DNA sequence analysis of the 51 patients with defective MMR revealed seven germline mutations-four in hMLH1 (two truncating and two missense) and three in hMSH2 (all truncating). A detailed family history was available for 225 of the 257 patients. Of the seven patients with germline mutations, only three had family histories consistent with hereditary nonpolyposis colorectal cancer. Of the remaining patients who had tumors with defective MMR, eight had somatic mutations in hMLH1. In addition, hypermethylation of the hMLH1 gene promoter was present in 37 (88%) of the 42 hMLH1-negative cases available for study and in all MSI-H tumors that showed loss of hMLH1 expression but no detectable hMLH1 mutations. Our results suggest that, although defective DNA MMR occurs in approximately 20% of unselected patients presenting for CRC resection, hereditary CRC due to mutations in the MMR pathway account for only a small proportion of patients. Of the 257 patients, only 5 (1.9%) appear to have unequivocal evidence of hereditary defects in MMR. The epigenetic (nonhereditary) mechanism of hMLH1 promoter hypermethylation appears to be responsible for the majority of the remaining patients whose tumors are characterized by defective DNA MMR.     

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants

The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.     

Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox

Genetic resistance to clinical mousepox (ectromelia virus) varies among inbred laboratory mice and is characterized by an effective natural killer (NK) response and the early onset of a strong CD8(+) cytotoxic T-lymphocyte (CTL) response in resistant mice. We have investigated the influence of virus-expressed mouse interleukin-4 (IL-4) on the cell-mediated response during infection. It was observed that expression of IL-4 by a thymidine kinase-positive ectromelia virus suppressed cytolytic responses of NK and CTL and the expression of gamma interferon by the latter. Genetically resistant mice infected with the IL-4-expressing virus developed symptoms of acute mousepox accompanied by high mortality, similar to the disease seen when genetically sensitive mice are infected with the virulent Moscow strain. Strikingly, infection of recently immunized genetically resistant mice with the virus expressing IL-4 also resulted in significant mortality due to fulminant mousepox. These data therefore suggest that virus-encoded IL-4 not only suppresses primary antiviral cell-mediated immune responses but also can inhibit the expression of immune memory responses.     

Isotopomer analysis using GC-MS

Knowledge of the complete isotopomer distribution represents the ultimate amount of information on the labeling pattern of a metabolite. One technique for measuring the isotopomer distributions is the analysis of the multiplet intensities arising from the 13C-13C couplings in NMR spectroscopy. While this technique has proven to be very valuable in the elucidation of labeling patterns of C2 and C3 units of various amino acids, fragments larger than C3 are very difficult to measure. Another technique, GC-MS, offers a unique possibility of analyzing fragments larger than C3 and GC-MS is therefore able to give information which is complementary to the information that can be obtained from NMR spectroscopy. In this work we have developed fast, simple, and robust GC-MS methods that can be used to gain information on the labeling patterns of the amino acids in a crude biomass hydrolysate. It is shown that a combination of information obtained from these analyses and information from the NMR spectroscopy is able to yield a much more complete picture of the isotopomer distributions of the amino acids than any of the two techniques alone. The GC-MS method was used for analyzing the labeling patterns of amino acids from a batch cultivation of Penicillium chrysogenum grown on fully labeled glucose. The data from this analysis showed no signs of any significant carbon isotope effects, and the measurements can therefore be used without corrections for metabolic flux analysis.     

Multiplex relative RT-PCR method for verification of differential gene expression

Differential display, suppression subtractive hybridization and other techniques for identification of differentially expressed genes produce fragments of cDNA from mRNAs whose differences in abundance must be verified. This report describes a relative multiplex RT-PCR assay that facilitates the analysis of large numbers of samples for differences in mRNA abundance without the use of radioactivity or blotting. The species of interest is co-amplified with 18S rRNA over a range of cycles followed by electrophoresis through ethidium bromide-agarose gels. Intensities of the bands of interest, normalized for 18S band intensities, are plotted as a function of cycle number. Regression equations fitted to the curves are used to calculate the number of cycles necessary for each sample's normalized signal to reach a threshold intensity. Differences between samples in the number of cycles required to reach that threshold reflect differences in the original abundances of those species. A comparison with results previously obtained using northern blots showed that relative differences as small as 20% and as large as an order of magnitude are accurately detected. The simplicity of the assay allows its routine application in both research and teaching laboratories.     

A nitrate-induced frq-less oscillator in Neurospora crassa

When nitrate is the only nitrogen source, Neurospora crassa's nitrate reductase (NR) shows endogenous oscillations in its nitrate reductase activity (NRA) on a circadian time scale. These NRA oscillations can be observed in darkness or continuous light conditions and also in a frq(9) mutant in which no functional FRQ protein is formed. Even in a white-collar-1 knockout mutant, NRA oscillations have been observed, although with a highly reduced amplitude. This indicates that the NRA oscillations are not a simple output rhythm of the white-collar-driven frq oscillator but may be generated by another oscillator that contains the nit-3 autoregulatory negative feedback loop as a part. In this negative feedback loop, a product in the reaction chain catalyzed by nitrate reductase, probably glutamine, induces repression of the nitrate reductase gene and thus downregulates its own production. This is the first example of an endogenous, nutritionally induced daily rhythm with known molecular components that is observed in the absence of an intact FRQ protein.