Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.     

Mapping and QTL analysis of horticultural traits in a narrow cross in cucumber (Cucumis sativus L.) using random-amplified polymorphic DNA markers

An 80-point genetic map [77 random-amplified polymorphic DNAs (RAPD), F (female sex expression), de (determinate), and ll (little leaf)] was constructed from a narrow cross in cucumber using the determinate, gynoecious, standard-sized leaf line G421 and the indeterminate, monoecious, little leaf line H-19. The map defined nine linkage groups and spanned ca. 600 cM with an average distance between markers of 8.4 ± 9.4 cM. The RAPD loci BC-551 and BC-592 were found to flank ll at 3.4 and 12.2 cM, respectively. The locus OP-L18-2 was linked (16 cM) to de, and the F locus was flanked by markers at 44 and 31 cM. One-hundred F3 families were used to identify quantitative trait loci (QTL) for sex expression, main stem length, number of lateral branches, days to anthesis, fruit number and weight, fruit length and diameter, and fruit length: diameter ratio in two replicated test locations (Wisconsin and Georgia). QTL on linkage group B explained major portions (R2 = ca. 2 to 74%) of the variation observed for sex expression, main stem length, lateral branch number, and fruit diameter (LOD = 2.1 to 29.8). Although ca. 62 to 74% of the variation for sex expression was associated with a putative QTL spanning the F locus (OP-AJ-2 to F and F to de), other regions (three) of the genome were important for the determination of sex in the F3 families examined depending upon environment. The number of genomic regions affecting main stem length (five) and number of lateral branches (three) coincided with expectations as determined by calculations of minimum number of genes in previous studies. Evaluation of QTL associated with several fruit number determinants of early, first-harvest yield demonstrating additive genetic variance (i.e., sex expression, main stem length, and number of laterals) suggests that marker-assisted selection may have utility for the development of determinate, multiple lateral branching germplasm suited for once-over mechanical harvesting in this population.     

Electrical and adaptive properties of rod photoreceptors in bufo marinus. II. Effects of cyclic nucleotides and protaglandins

Substances known to alter cyclic nucleotide levels in cells were applied to the isolated toad retina and effects on rod electrical and adaptive behavior were studied. The retina was continually superfused in control ringer’s or ringer’s containing one or a combination of drugs, and rod activity was recorded intracellularly. Superfusion with cGMP, Bu(2)GMP, isobutylmethylxanthine (IBMX; a phosphodiesterase inhibitor), or PGF(2α) (a prostaglandin) caused effects in rods that closely match those observed when extracellular Ca(2+) levels were lowered. For example, short exposures (up to 6 min) of the retina to these substances caused depolarization of the membrane potential, increase in response amplitudes, and some changes in waveform; but under dark-adapted or partially light-adapted conditions receptor sensitivity was virtually unaffected. That is, the position of the V-log I curve on the intensity axis was determined by the prevailing light level, not by drug level. These drugs, like lowered extracellular Ca(2+), also decreased the period of receptor saturation after a bright-adapting flash, resulting in an acceleration of the onset of membrane and sensitivity recovery during dark adaptation.

Long-term (6-15 min) exposure of a dark-adapted retina to 5 mM IBMX or a combination of IBMX and cGMP caused a loss of response amplitude and a desensitization of the rods that was similar to that observed in rods after a long-term low Ca(2+) (10(-9)M) treatment. Application of high (3.2 mM) Ca(2+) to the retina blocked the effects of applied Bu(2)cGMP. PGE(1) superfusion mimicked the effects of increasing extracellular Ca(2+). The results show that increased cGMP and lowered Ca(2+) produce similar alterations in the electrical activity of rods. These findings suggest that Ca(2+) and cGMP are interrelated messengers. We speculate that low Ca(2+) may lead to increased intracellular cGMP, and/or that applied cGMP, and/or that applied cGMP may lower cytosol Ca(2+), perhaps by stimulating Ca(2+)- ATPase pumps in the outer segment.

     

Stent implantation in acute myocardial infarction using a heparin-coated stent: a pilot study as a preamble to a randomized trial comparing balloon angioplasty and stenting

Preliminary experience with primary stenting in myocardial infarction has suggested a greater benefit in clinical outcome than has been obtained with direct balloon angioplasty. However, subacute thrombosis (SAT) remains a limitation for this new mode of therapy. In the BENESTENT II Pilot and main trials, the incidence of SAT with the heparin-coated Palmaz-Schatz stent was only 0.15%. Therefore, as a preamble to a large randomized trial, the feasibility and safety of the use of the Heparin-Coated Palmaz-Schatz trade mark Stent in Acute Myocardial Infarction (AMI) was tested in 101 patients enrolled between April and September 1996 in 18 clinical centres. In 101 stent-eligible AMI patients, as dictated by protocol, a heparin-coated stent was implanted. The primary objectives were to determine the in-hospital incidence of major adverse cardiac events (MACE: death, MI, target lesion revascularization) and bleeding complications, while the secondary objectives were the procedural success rate and the MACE, the restenosis and reocclusion rates at 6.5 months. Stent implantation (n 3 129 stents) was successful in 97 patients of the 101 who were included in this trial. During their hospital stay, two patients died and no patient experienced re-infarction, ischaemia prompting re-PTCA or CABG. Four patients suffered a bleeding complication, three major and one minor, of whom three required surgical repair. At 210 days follow-up, 81% of the patients were event free. At 6.5 months restenosis was documented in 18% of the 88 patients who underwent follow-up angiography, including three total occlusions. The results, both with respect to QCA and the occurrence of MACE, compare favourably with studies using elective stenting in both stable and unstable angina patients. As a result of this pilot study, a large randomized trial comparing direct balloon angioplasty with direct stenting in 900 patients with AMI was initiated in December 1996.     

Genetic conflict outweighs heterogametic incompatibility in the mouse hybrid zone?

Background  

The Mus musculus musculus/M. m. domesticus contact zone in Europe is characterised by sharp frequency discontinuities for sex chromosome markers at the centre of wider clines in allozyme frequencies.     

Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled-coil mating pair formation protein TrhB

Assemblies of plasmid-encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane-associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two-hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27-encoded proteins TraG and TrhB interact. This interaction was verified through a co-immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline-rich domain from amino acids 135-173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far-Western, co-immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled-coil region at residues 77-124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.     

Intercellular communication via intracellular calcium oscillations

In this letter, we present the results of a simple model for intercellular communication via calcium oscillations, motivated in part by a recent experimental study. The model describes two cells (a "donor" and "sensor") whose intracellular dynamics involve a calcium-induced, calcium release process. The cells are coupled by assuming that the input of the sensor cell is proportional to the output of the donor cell. As one varies the frequency of calcium oscillations of the donor cell, the sensor cell passes through a sequence of N : M phase-locked regimes and exhibits a "Devil's staircase" behavior. Such a phase-locked response has been seen experimentally in pulsatile stimulation of single cells. We also study a stochastic version of the coupled two-cell model. We find that phase locking holds for realistic choices for the cell volume.     

Stochastic effects in intercellular calcium spiking in hepatocytes

We carry out a Monte Carlo simulation of stochastic effects for two models of intercellular calcium wave propagation in rat hepatocytes. Both models involve gap junction diffusion by a second messenger. We find that, in general, the stochastic effects improve agreement with experiment, for a reasonable choice of model parameters. Both stochastic models exhibit baseline fluctuations and variations in the peak heights of Ca(2+). In addition, we find for one model that there is a distribution of latency times, rather than a single latency time, with a distribution width which is comparable to the experimental observation of spike widths. We also find for the other model with low gap junction diffusion that it is possible for cell multiplets to oscillate independently initially, but to subsequently become synchronized.