Laser microdissection expression profiling of marginal edges of colorectal tumours reveals evidence of increased lactate metabolism in the aggressive phenotype.

BACKGROUND: The morphology of the invasive margin in colorectal cancer can be described as either pushing or infiltrative. These phenotypes carry prognostic significance, particularly in node negative disease, and provide an excellent model for the study of invasive behaviour in vivo. METHODS: The marginal edges of 16 stage-matched tumours exhibiting these contrasting growth patterns were microdissected. The extracted mRNA was amplified and hybridised to a 9546 feature oligonucleotide array. Selected differentials were validated using real-time polymerase chain reaction and the protein product was interrogated by using immunohistochemistry. RESULTS: After stringent quality control and filtering of data generated, 39 genes were identified as being significantly differentially expressed between the two types of marginal edge. Several genes involved in cellular metabolism were identified as differentials including lactate dehydrogenase B (LDHB) and modulators of glucose transport. CONCLUSIONS: The LDH expression profile differs between the invasive phenotypes. A hypothesis is proposed in which altered metabolism is a cause of contrasting invasive behaviour independent of the hypoxia-inducible factor mediated hypoxic response, consistent with the Warburg phenomenon.

Temporal expression profiling of the uterine luminal epithelium of the pseudo-pregnant mouse suggests receptivity to the fertilized egg is associated with complex transcriptional changes.

BACKGROUND: The molecular basis of changes underlying the altered sensitivity of the uterine luminal epithelium (LE) to the embryo over the peri-implantation period is not fully understood. METHODS: Microarray analysis was performed on purified LE isolated from the pseudo-pregnant mouse uterus at 12-h intervals from pre-receptivity through the implantation window to refractoriness. The aim was to identify genes whose expression changes in the LE during this period. RESULTS: A total of 447 transcripts were identified whose abundance changed more than 2-fold in the LE but which did not change in the underlying stroma (S) and glands. Six major patterns of changing expression were noted. Of the 447 genes, 140 were expressed in LE at least 15-fold higher than in S and glandular epithelium (GE) (101 of these more than 20-fold). Detailed spatiotemporal expression profiles were derived for several genes previously implicated in implantation (including Edg7, Ptgs1, Pla2g4a and Alox15). CONCLUSIONS: Functional changes in LE receptivity are characterized by changing constellations of gene expression. Pre-receptivity has a different molecular footprint to refractoriness. Because we have used the pseudo-pregnant mouse model, these changes are driven solely by endocrine signals rather than events downstream of embryo attachment. Some of these genes have been described in previous microarray studies on endometrium, but for the majority, this is the first time they have been implicated in implantation. The 140 genes enriched in the LE greatly expand the list of epithelial markers and provide many novel candidates for further studies to identify genes playing important roles in receptivity and embryo attachment.

Identification of genes regulated by leukemia-inhibitory factor in the mouse uterus at the time of implantation.

The endometrium is prepared for implantation by the actions of estradiol (E2) and progesterone (P4). In mice the luminal epithelium (LE) only becomes fully receptive to the attaching blastocyst in response to the nidatory estrogen surge on d 4 of pregnancy. The cytokine leukemia-inhibitory factor (LIF) is rapidly induced by nidatory estrogen and has been shown to be the primary mediator of its action. Implantation fails in the absence of LIF, and injection of LIF on d 4 of pregnancy can substitute for the nidatory estrogen. In this study, we sought to identify genes regulated by LIF in the uterine epithelium. We used oligonucleotide microarrays to compare the transcript profiles of paired uterine horns from LIF-deficient MF1 mice after intraluminal injection of LIF or PBS on d 4 of pseudopregnancy. IGF-binding protein 3 was identified as a gene up-regulated by LIF; this was confirmed by RT-PCR. In situ hybridization showed that the primary site of IGF-binding protein 3 expression is the luminal epithelium (LE), the known site of LIF action in the uterus. We identified two other genes: amphiregulin and immune response gene-1, the expression of which were also up-regulated by LIF. Immune response gene 1 has recently been shown to be essential for implantation. Expression of all three of these genes in the LE is known to be regulated by P4. The expression of osteoblast-specific factor 2 and leukocyte 12/15 lipoxygenase, which are also expressed in LE under the control of P4, were not increased by LIF. This suggests that one of the actions of LIF on LE may be to enhance the expression of a subset of P4-regulated genes.

An analysis using DNA microarray of the time course of gene expression during syncytialization of a human placental cell line (BeWo).

Placental trophoblast syncytialization is a unique biological process. We have studied the time course of this process using DNA microarray in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin). Total RNA was extracted from BeWo cells and labelled-cRNA target was then hybridized to a specific oligonucleotide probe set containing probes to over 12?000 human transcripts. Detectable levels of signal were found on average for 44 per cent of the total number of genes assayed. The correlation coefficient for the level of expression of independent replicates was #10878;0.99. The mRNA expression profile of specific genes analysed by microarray correlated quantitatively well with that analysed by reverse transcription-polymerase chain reaction and with protein secretion. In the absence of forskolin there are relatively few changes in gene expression (reaching a threshold of two fold); in the presence of forskolin there are a substantial number of changes. By clustering the patterns of altered gene expression at least ten groups could be extracted. Seven of these clusters involved increased gene expression and three decreased expression. Each cluster has been categorized by gene ontology (confining the analysis to genes with 'known' function). Among the genes with increased expression following forskolin treatment were many required for cellular communication (such as placental specific peptide hormones) and metabolism (such as cholesterol side chain cleavage enzyme). Several genes known to be involved in cell adhesion and fusion have markedly changed expression levels very early following forskolin exposure, thus preceding morphological fusion of BeWo cells. Further analysis of this data and expression profiling in general will be able to contribute to understanding the functional basis for the formation of the placental syncytiotrophoblast.

Global amplification of mRNA by template-switching PCR: linearity and application to microarray analysis.

Conventional approaches to target labelling for expression microarray analysis typically require relatively large amounts of total RNA, a serious limitation when the sample available is small. Here we explore the cycle-dependent amplification characteristics of Template-Switching PCR and validate its use for microarray target labelling. TS-PCR identifies up to 80% of the differentially expressed genes identified by direct labelling using 30-fold less input RNA for the amplification, with the equivalent of 1000-fold less starting material being used for each hybridisation. Moreover, the sensitivity of microarray experiments is increased considerably, allowing the identification of differentially expressed transcripts below the level of detection using targets prepared by direct labelling. We have also validated the fidelity of amplification and show that the amplified material faithfully represents the starting mRNA population. This method outperforms conventional labelling strategies, not only in terms of sensitivity and the identification of differentially expressed genes, but it is also faster and less labour intensive than other amplification protocols.

Rhythm generation in monkey motor cortex explored using pyramidal tract stimulation.

We investigated whether stimulation of the pyramidal tract (PT) could reset the phase of 15-30 Hz beta oscillations observed in the macaque motor cortex. We recorded local field potentials (LFPs) and multiple single-unit activity from two conscious macaque monkeys performing a precision grip task. EMG activity was also recorded from the second animal. Single PT stimuli were delivered during the hold period of the task, when oscillations in the LFP were most prominent. Stimulus-triggered averaging of the LFP showed a phase-locked oscillatory response to PT stimulation. Frequency domain analysis revealed two components within the response: a 15-30 Hz component, which represented resetting of on-going beta rhythms, and a lower frequency 10 Hz response. Only the higher frequency could be observed in the EMG activity, at stronger stimulus intensities than were required for resetting the cortical rhythm. Stimulation of the PT during movement elicited a greatly reduced oscillatory response. Analysis of single-unit discharge confirmed that PT stimulation was capable of resetting periodic activity in motor cortex. The firing patterns of pyramidal tract neurones (PTNs) and unidentified neurones exhibited successive cycles of suppression and facilitation, time locked to the stimulus. We conclude that PTN activity directly influences the generation of the 15-30 Hz rhythm. These PTNs facilitate EMG activity in upper limb muscles, contributing to corticomuscular coherence at this same frequency. Since the earliest oscillatory effect observed following stimulation was a suppression of firing, we speculate that inhibitory feedback may be the key mechanism generating such oscillations in the motor cortex.

Transducer models of head-centred motion perception.

By adding retinal and pursuit eye-movement velocity one can determine the motion of an object with respect to the head. It would seem likely that the visual system carries out a similar computation by summing extra-retinal, eye-velocity signals with retinal motion signals. Perceived head-centred motion may therefore be determined by differences in the way these signals encode speed. For example, if extra-retinal signals provide the lower estimate of speed then moving objects will appear slower when pursued (Aubert-Fleischl phenomenon) and stationary objects will move opposite to an eye movement (Filehne illusion). Most previous work proposes that these illusions exist because retinal signals encode retinal motion accurately while extra-retinal signals under-estimate eye speed. A more general model is presented in which both signals could be in error. Two types of input/output speed relationship are examined. The first uses linear speed transducers and the second non-linear speed transducers, the latter based on power laws. It is shown that studies of the Aubert-Fleischl phenomenon and Filehne illusion reveal the gain ratio or power ratio alone. We also consider general velocity-matching and show that in theory matching functions are limited by gain ratio in the linear case. However, in the non-linear case individual transducer shapes are revealed albeit up to an unknown scaling factor. The experiments show that the Aubert-Fleischl phenomenon and Filehne illusion are adequately described by linear speed transducers with a gain ratio less than one. For some observers, this is also the case in general velocity-matching experiments. For other observers, however, behaviour is non-linear and, according to the transducer model, indicates the existence of expansive non-linearities in speed encoding. This surprising result is discussed in relation to other theories of head-centred motion perception and the possible strategies some observers might adopt when judging stimulus motion during an eye movement.

Extraretinal and retinal amplitude and phase errors during Filehne illusion and path perception.

Pursuit eye movements give rise to retinal motion. To judge stimulus motion relative to the head, the visual system must correct for the eye movement by using an extraretinal, eye-velocity signal. Such correction is important in a variety of motion estimation tasks including judgments of object motion relative to the head and judgments of self-motion direction from optic flow. The Filehne illusion (where a stationary object appears to move opposite to the pursuit) results from a mismatch between retinal and extraretinal speed estimates. A mismatch in timing could also exist. Speed and timing errors were investigated using sinusoidal pursuit eye movements. We describe a new illusion--the slalom illusion--in which the perceived direction of self-motion oscillates left and right when the eyes move sinusoidally. A linear model is presented that determines the gain ratio and phase difference of extraretinal and retinal signals accompanying the Filehne and slalom illusions. The speed mismatch and timing differences were measured in the Filehne and self-motion situations using a motion-nulling procedure. Timing errors were very small for the Filehne and slalom illusions. However, the ratios of extraretinal to retinal gain were consistently less than 1, so both illusions are the consequence of a mismatch between estimates of retinal and extraretinal speed. The relevance of the results for recovering the direction of self-motion during pursuit eye movements is discussed.

Unequal retinal and extra-retinal motion signals produce different perceived slants of moving surfaces.

Eye movements introduce retinal motion to the image and so affect motion cues to depth. For instance, the slant of a plane moving at right-angles to the observer is specified by translation and a component of relative motion such as shear. To a close approximation, the translation disappears from the image when the eye tracks the surface accurately with a pursuit eye movement. However, both translation and relative-motion components are needed to estimate slant accurately and unambiguously. During pursuit, therefore, an extra-retinal estimate of translation must be used by the observer to estimate surface slant. Extra-retinal and retinal estimates of translation speed are known to differ: a classic Aubert-Fleischl phenomenon was found for our stimuli. The decrease in perceived speed during pursuit predicts a corresponding increase in perceived slant when the eye tracks the surface. This was confirmed by comparing perceived slant in pursuit and eye-stationary conditions using slant-matching and slant-estimation techniques. Moreover, the increase in perceived slant could be quantified solely on the basis of the perceived-speed data. We found no evidence that relative-motion estimates change between the two eye-movement conditions. A final experiment showed that perceived slant decreases when a fixed retinal shear is viewed with increasing pursuit speed, as predicted by the model. The implication of the results for recovering metric depth estimates from motion-based cues is discussed.

Adenosine receptor expression and function in rat striatal cholinergic interneurons.

Cholinergic neurons were identified in rat striatal slices by their size, membrane properties, sensitivity to the NK(1) receptor agonist (Sar(9), Met(O(2))(11)) Substance P, and expression of choline acetyltransferase mRNA. A(1) receptor mRNA was detected in 60% of the neurons analysed, and A(2A) receptor mRNA in 67% (n=15). The A(1) receptor agonist R-N(6)-(2-phenylisopropyl)adenosine (R-PIA) hyperpolarized cholinergic neurons in a concentration dependent manner sensitive to the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 100 nM). In dual stimulus experiments, the A(2A) receptor antagonist 8-(3-chlorostyryl)caffeine (CSC, 500 nM) decreased release of [(3)H]-acetylcholine from striatal slices (S2/S1 0.78+/-0.07 versus 0.95+/-0.05 in control), as did adenosine deaminase (S2/S1 ratio 0.69+/-0.05), whereas the A(1) receptor antagonist DPCPX (100 nM) had no effect (S2/S1 1.05+/-0.14). In the presence of adenosine deaminase the adenosine A(2A) receptor agonist 2-p-((carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadeno sin e (CGS21680, 10 nM) increased release (S2/S1 ratio 1.03+/-0.05 versus 0.88+/-0.05 in control), an effect blocked by the antagonist CSC (500 nM, S2/S1 0.68+/-0.05, versus 0.73+/-0.08 with CSC alone). The combined superfusion of bicuculline (10 microM), saclofen (1 microM) and naloxone (10 microM) had no effect on the stimulation by CGS21680 (S2/S1 ratio 0.99+/-0.04). The A(1) receptor agonist R-PIA (100 nM) inhibited the release of [(3)H]-acetylcholine (S2/S1 ratio 0.70+/-0.03), an effect blocked by DPCPX (S2/S1 ratio 1.06+/-0.07). It is concluded that both A(1) and A(2A) receptors are expressed on striatal cholinergic neurons where they are functionally active.

Improved method for detecting differentially expressed genes using cDNA indexing.

In cDNA indexing, differentially expressed genes are identified by the display of specific, corresponding subsets of cDNA. Subdivision of the cDNA population is achieved by the sequence-specific ligation of adapters to the overhangs created by class IIS restriction enzymes. However, inadequate specificity of ligation leads to redundancy between different adapter subsets. We evaluate the incidence of mismatches between adapters and class IIS restriction fragments during ligation and describe a modified set of conditions that improves ligation specificity. The improved protocol reduces redundancy between amplified cDNA subsets, which leads to a lower number of bands per lane of the differential display gel, and therefore simplifies analysis. We confirm the validity of this revised protocol by identifying five differentially expressed genes in mouse duodenum and ileum.

Characterisation of a novel murine intestinal serine protease, DISP.

A putative novel murine serine protease, DISP, was identified by cDNA indexing and shown to be expressed primarily in distal gut. FISH analysis showed it to be localised to mouse chromosome 17A3. A possible human homologue for DISP has been identified. DISP is a novel member of clan SA/family S1 of the serine proteases, at present of unknown function.

Correlating physiology with gene expression in striatal cholinergic neurones.

The expression of 34 transmitter-related genes has been examined in the cholinergic neurones of rat striatal brain slices, with the aim of correlating gene expression with functional activity. The mRNAs encoding types I, II/IIA, and III alpha subunits of the voltage-sensitive sodium channels were detected, suggesting the presence of these three types of sodium channel. Similarly, mRNAs encoding all four alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptor subunits and the NR1 and NR2A, 2B, and 2D subunits of the NMDA-type glutamate receptors were detected, suggesting that various combinations of these subunits mediate the cellular response to synaptically released glutamate. Other mRNAs detected included the NK1 and NK3 tachykinin receptors, all four known adenosine receptors, and the GABA-synthesising enzyme glutamate decarboxylase. Subpopulations of these cholinergic neurones have been identified on the basis of the expression of the NK3 tachykinin receptor in 5% and the trkC neurotrophin receptor in 12% of the cells investigated.

Analysis of gene expression in single cells.

A cell's structural and functional characteristics are dependent on the specific complement of genes it expresses. The ability to study and compare gene usage at the cellular level will therefore provide valuable insights into cell physiology. Such analyses are complicated by problems associated with sample collection, sample size and the limited sensitivity of expression assays. Advances have been made in approaches to the collection of cellular material and the performance of single-cell gene expression analysis. Recent development in global amplification of mRNA may soon permit expression analyses of single cells to be performed on DNA microarrays.

The human family of Deafness/Dystonia peptide (DDP) related mitochondrial import proteins.

The gene responsible for the human genetic neurodegenerative disorder DFN-1/MTS encodes a small protein known as deafness/dystonia peptide (DDP). It bears a strong resemblance to a recently characterized set of zinc-binding yeast proteins (Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p) that are implicated in the import of a class of transmembrane carrier proteins from the cytoplasm to the mitochondrial inner membrane. We describe here the human complement of DDP/Tim-like proteins and establish the likely orthologous relationships between sequences from human, yeast, and other organisms. We also describe the expression patterns and chromosomal locations of their genes, which are candidate loci for autosomal recessive neurodegenerative disorders.

Path perception and Filehne illusion compared: model and data.

Pursuit eye movements introduce retinal motion that complicates the recovery of self-motion from retinal flow. An extra-retinal, eye-velocity signal could be used to aid estimation of the observer's path, perhaps by converting retino-centric into head-centric motion. This conversion is apparently not precise because we often misperceive head-centric object velocity: in the Filehne illusion, for example, a stationary object appears to move in the opposite direction to the eye movement. Similar errors should be expected when extra-retinal, eye-velocity signals are used in self-motion tasks. However, most self-motion studies conclude that path direction is recovered quite accurately. Path perception and the Filehne illusion were therefore compared directly in order to examine the apparent discrepancy. A nulling technique determined the velocity of simulated eye rotation that cancelled the perceived curvature of the path or, in a Filehne condition, the perceived rotation of the ground-plane stimulus. In either case, observers typically set the simulated eye rotation to be a fixed proportion of the actual eye pursuit made. No differences were found between path perception and Filehne illusion. The apparent inaccuracy of path perception during a real eye movement was confirmed in a second experiment, using a standard 'mouse-pointing' technique. The experiments provide support for a model of head-centric motion perception based on extra-retinal and retinal signals that are linearly related to pursuit and retinal speed, respectively.

Different mechanisms underlie three inhibitory phenomena in cat area 17.

Recently, it has been proposed that all suppressive phenomena observed in the primary visual cortex (V1) are mediated by a single mechanism, involving inhibition by pools of neurons, which, between them, represent a wide range of stimulus specificities. The strength of such inhibition would depend on the stimulus that produces it (particularly its contrast) rather than on the firing rate of the inhibited cell. We tested this hypothesis by measuring contrast-response functions (CRFs) of neurons in cat V1 for stimulation of the classical receptive field of the dominant eye with an optimal grating alone, and in the presence of inhibition caused by (1) a superimposed orthogonal grating (cross-orientation inhibition); (2) a surrounding iso-oriented grating (surround inhibition); and (3) an orthogonal grating in the other eye (interocular suppression). We fitted hyperbolic ratio functions and found that the effect of cross-orientation inhibition was best described as a rightward shift of the CRF ('contrast-gain control'), while surround inhibition and interocular suppression were primarily characterised as downward shifts of the CRF ('response-gain control'). However, the latter also showed a component of contrast-gain control. The two modes of suppression were differently distributed between the layers of cortex. Response-gain control prevailed in layer 4, whereas cells in layers 2/3, 5 and 6 mainly showed contrast-gain control. As in human observers, surround gratings caused suppression when the central grating was of high contrast, but in over a third of the cells tested, enhanced responses for low-contrast central stimuli, hence actually decreasing threshold contrast.

Expression profiling of single cells using 3 prime end amplification (TPEA) PCR.

The ability to relate the physiological status of individual cells to the complement of genes they express is limited by current methodological approaches for performing these analyses. We report here the development of a robust and reproducible method for amplifying 3' sequences of mRNA derived from single cells and demonstrate that the amplified cDNA, derived from individual human lymphoblastoma cells, can be used for the expression profiling of up to 40 different genes per cell. In addition, we show that 3 prime end amplification (TPEA) PCR can be used to enable the detection of both high and low abundance mRNA species in samples harvested from live neurons in rat brain slices. This procedure will facilitate the study of complex tissue function at the cellular level.

Identification of an ATP-sensitive potassium channel current in rat striatal cholinergic interneurones.

1. Whole-cell patch-clamp recordings were made from rat striatal cholinergic interneurones in slices of brain tissue in vitro. In the absence of ATP in the electrode solution, these neurones were found to gradually hyperpolarize through the induction of an outward current at -60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide and by the photorelease of caged ATP within neurones. 2. This ATP-sensitive outward current was not observed when 2 mM ATP was present in the electrode solution. Under these conditions, 500 microM diazoxide was found to induce an outward current that was blocked by tolbutamide. 3. Using permeabilized patch recordings, neurones were shown to hyperpolarize in response to glucose deprivation or metabolic poisoning with sodium azide (NaN3). The resultant hyperpolarization was blocked by tolbutamide. 4. In cell-attached recordings, metabolic inhibition with 1 mM NaN3 revealed the presence of a tolbutamide-sensitive channel exhibiting a unitary conductance of 44.1 pS. 5. Reverse transcription followed by the polymerase chain reaction using cytoplasm from single cholinergic interneurones demonstrated the expression of the ATP-sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2. 6. It is concluded that cholinergic interneurones within the rat striatum exhibit a KATP channel current and that this channel is formed from Kir6.1 and SUR1 subunits.