The serotonin transporter intronic VNTR enhancer correlated with a predisposition to affective disorders has distinct regulatory elements within the domain based on the primary DNA sequence of the repeat unit.

We have demonstrated that a variable number tandem repeat domain (VNTR) within intron 2 of the serotonin transporter gene is a transcriptional regulatory domain which is potentially correlated with a predisposition to affective disorders and other behavioural conditions. This correlation based on copy number of the VNTR alone (nine, 10 or 12 copies of 16/17 base-pair element) has been controversial and not reproduced in all studies. We demonstrate that individual repeat elements within the VNTR domain differ in their enhancer activity in an embryonic stem cell model. This has implications for both the mechanism by which these VNTRs are correlated with the progression of the disease and suggests that clinical analysis should now be extended to correlate sequence variation within the VNTR with the disorder. The latter may resolve some of the conflicting data published to date.

Tachykinin expression in cartilage and function in human articular chondrocyte mechanotransduction.

OBJECTIVE: To assess whether substance P and the corresponding neurokinin 1 (NK1) receptor are expressed in human articular cartilage, and whether these molecules have a role in chondrocyte mechanotransduction. METHODS: Transgenic studies, immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction were used to assess the expression of the preprotachykinin (PPT) gene, substance P, and NK1 in developing mice, in adult human articular cartilage, and in human chondrocytes in culture. Chondrocytes obtained from PPT knockout mice and human articular chondrocytes were mechanically stimulated in the presence or absence of inhibitors of substance P signaling, and cell membrane potentials or relative levels of aggrecan messenger RNA (mRNA) were measured. RESULTS: Replacing a region of the PPT gene transcriptional site that contains a dominant repressor of the proximal promoter activity with the constitutive minimal promoter of the human beta-globin promoter allowed expression of a marker gene in areas of chondrogenesis during mouse development and in adult chondrocytes grown in culture. Adult human articular chondrocytes expressed endogenous PPT mRNA, substance P, and the corresponding NK1 receptor in vivo and in vitro. Blockade of substance P signaling by a chemical antagonist to the NK1 receptor inhibited chondrocyte responses to mechanical stimulation. CONCLUSION: Substance P is expressed in human articular cartilage and is involved in chondrocyte mechanotransduction via the NK1 receptor in an autocrine and paracrine manner. This suggests that substance P and the NK1 receptor have roles in the maintenance of articular cartilage structure and function that were previously unrecognized.

Molecular models to analyse preprotachykinin-A expression and function.

Towards an understanding of the mechanisms controlling Preprotachykinin A (PPT) expression we have generated a variety of molecular models to determine the mechanisms regulating both the tissue-specific and stimulus-inducible expression of the PPT gene. The approaches used include transgenic and virus vector models complementing biochemical analysis of promoter interactions with transcription factors. We have identified and characterised a yeast artificial chromosome (YAC) containing the human PPT gene and generated transgenic mouse lines containing multiple copies of this chromosome on a normal mouse genetic background. This resulted in a pattern of expression in the nervous system remarkably similar to that reported for PPT mRNA in rodents. In addition, this transgenic model has been constructed in such a manner to allow for over expression of tachykinins based on the number of extra alleles in the transgenic mouse. These animals allow us to further examine the function of the tachykinins and acts as a useful complement to existing PPT ablated mice. In vitro we have introduced the proximal PPT promoter in reporter gene constructs into adult neurones in both DRG and the CNS by an adenoassociated virus (AAV) vector or by biolistic transfection respectively. Using the AAV vector we have demonstrated that the proximal promoter can mediate the effects of NGF in adult rat DRG. These models allow us to delineate transcriptional domains involved in the physiological and pathological expression of the PPT gene.

A role for Octamer binding protein motifs in the regulation of the proximal preprotachykinin-A promoter.

We have recently developed cell line models that express the endogenous rat preprotachykinin-A (rPPT) gene and support reporter gene expression directed by the rPPT promoter. These are the neuronal derived cell line NF2C and the pancreatic cell lines RINm5F and a derivative RIN-1027-B2. Reporter gene activity in these cell lines is similar to that observed in primary cultures of rat dorsal root ganglion neurons. Analysis of reporter gene expression supported by various fragments of the rPPT promoter demonstrated that although -865 to +92 supported expression, addition of fragments between +92 and +500 led to repression of expression. Two previously defined octamer binding motifs, present both 5' and 3' of the major transcriptional start site, have been postulated to be potential enhancers of rPPT activity and we have now used these cell lines to determine the role of these regions in the rPPT promoter. Site specific mutagenesis of these elements has shown not only that both sites are potential enhancers of gene expression but also that the 3' element binds multiple factors, of which at least one has repressor function. Binding of this repressor protein over or adjacent to the 3' octamer binding protein site leads to the observed repression of promoter activity in the -865 to +500 construct relative to the to -865 to +92 the fragment.

E-box motifs within the human vasopressin gene promoter contribute to a major enhancer in small-cell lung cancer.

[Arginine]vasopressin (AVP) is a neuropeptide physiologically synthesized in the hypothalamus but pathologically expressed by small-cell lung cancer (SCLC). A minimal 65 bp AVP promoter can restrict basal activity to SCLC in vitro, but a 199 bp fragment directs 5-fold higher expression in SCLC [Coulson, Stanley and Woll (1999) Br. J. Cancer 80, 1935-1944]. Several predicted E-box motifs occur within the 199 bp fragment, and we now describe an enhancer which contributes to AVP promoter tumour-specificity in some cell lines. The deletion of two adjacent E-boxes (-157 to -131) resulted in an approx. 70% loss of reporter gene expression in a SCLC line (Lu-165) with high endogenous AVP production. Using a series of AVP promoter deletion constructs and site-directed mutagenesis, we show that both these E-box sites were required for enhancer function, whereas mutation of an adjacent AP-1 site had no effect on the promoter activity. Electrophoretic-mobility-shift analysis indicated that, although both the predicted E-box motifs bound specific complexes, only one appeared to function as a strong E-box which binds basic helix-loop-helix (bHLH) factors. This motif formed a complex in lung tumour-cell extracts, which was particularly strongly bound in Lu-165, and was competed for by a characterized E-box motif from the preprotachykinin A promoter. Antibody supershifts indicate that this complex is a heterodimer of upstream stimulatory factor (USF)-1 and USF-2. Non-bHLH complexes weakly bound the second potential E-box motif in a SCLC-specific manner. These complexes were not recognized by the bHLH antibodies and remain unidentified; however, they were detected in seven of eight SCLC cell lines and not in four control lines. We postulate that there is a co-operative and complex interaction between an E-box and an adjacent site constituting a SCLC-specific enhancer within the AVP proximal promoter.

Arginine vasopressin promoter regulation is mediated by a neuron-restrictive silencer element in small cell lung cancer.

Arginine vasopressin (AVP) is often expressed in small cell lung cancer (SCLC), and a 65-bp AVP minimal promoter fragment is sufficient to restrict activity to SCLC in vitro. We now describe a motif with homology to the neuron-restrictive silencer element (NRSE) within this fragment. Electrophoretic mobility shift analysis demonstrated that multiple specific complexes are bound by this motif. These complexes are cross-competed with a characterized SCG10 NRSE probe and do not bind to the AVP probe with a specific mutation in the NRSE. The complexes vary in mobility between lung tumor cell lines, showing different levels of AVP expression, and some are differentially bound in SCLC. Overexpression of a neuron-restrictive silencer factor expression construct can silence reporter gene expression supported by the AVP promoter in SCLC, although this was dependent on both the level of endogenous AVP expression in the cells and putative enhancer elements in larger promoter constructs. Activation of the proximal AVP promoter in SCLC is therefore proposed to, at least partially, rely on modulation of normal repressor activity at the NRSE.

Behavioural changes in the rat following infection with varicella-zoster virus.

Following the establishment of a chronic varicella-zoster virus infection in the rat, behavioural allodynia and hyperalgesia were observed in the injected, but not the contralateral hind limb up to 33 days post-infection. This model may prove useful in investigating mechanisms involved in the establishment of post-herpetic neuralgia.

An intronic polymorphic domain often associated with susceptibility to affective disorders has allele dependent differential enhancer activity in embryonic stem cells.

Variable number tandem repeats (VNTR) within non-coding regions of a number of genes have been correlated with susceptibility to various disease states. In particular, a VNTR polymorphism of a 16 or 17 bp element within intron 2 of the human serotonin transporter gene has been correlated with a predisposition to affective disorders. We have demonstrated that this region will support differential levels of reporter gene expression in differentiating embryonic stem cells, this being dependent on the presence of 10 or 12 copies of the repeat. The VNTR domain can therefore act as a transcriptional regulator, a property which potentially contributes to disease susceptibility.

Novel cell lines for the analysis of preprotachykinin A gene expression identify a repressor domain 3' of the major transcriptional start site.

Until now, no clonal cells have been identified that support the expression of a marker gene expressed from the rat preprotachykinin A (rPPT) promoter. We have analysed recently available cell lines that are candidates for supporting reporter gene expression directed by the rPPT promoter. These are the neuronal-derived cell line NF2C and the pancreatic cell lines RINm5F and a derivative RIN-1027-B2. The NF2C line was derived from the brain homogenate of a transgenic animal in which a temperature-sensitive simian virus 40 large T antigen was expressed from a neurofilament promoter. All three lines are able to support expression of a reporter gene directed by a fragment of the 5' rPPT promoter. Analysis of reporter gene expression supported by various fragments of the rPPT promoter demonstrated that although -865 to +92 bp supported expression, the addition of fragments between +92 and +447 bp led to repression of expression. Subsequent analysis of reporter gene constructs microinjected into primary cultures of dorsal root ganglion neurons (DRG) confirmed the existence of this repressor domain. This repression could be relieved totally in both RIN cell lines and partly in NF2C cells by mutating residues between +373 and +396 bp. This indicates that these cell lines support PPT promoter activity similar to that observed in DRG and determines a novel repressor domain within the promoter.

An intronic domain within the rat preprotachykinin-A gene containing a CCCT repetitive motif acts as an enhancer in differentiating embryonic stem cells.

Previous attempts by several groups to clone fragments containing intron 2 of the rat preprotachykinin-A gene have generated deletions of various sizes. We have determined that these deletions occur within a specific region of the intron spanning a CCCT tandem repeat domain. We show that this intronic domain is able to support reporter gene expression in mouse embryonic stem (ES) cells that have been induced to differentiate but not in undifferentiated ES cells. No significant expression was observed in the HeLa clonal cell line. This demonstrates that this intron 2 domain is a highly restrictive enhancer and may be associated with differentiation.

Identification of a novel multifunctional structural domain in the herpes simplex virus type 1 genome: implications for virus latency.

A domain, previously termed RE1, exists within the herpes simplex virus type 1 genome potentially influencing expression of immediate early genes and the latency associated transcripts. This domain consists of 10 tandem copies of a CT-rich sequence. We demonstrate that this domain binds multiple host-cell factors that may allow RE1 to act either as a transcriptional regulator and/or to affect nucleosomal and DNA structure in the latent genome.

A role for the octamer-binding protein in preprotachykinin-A gene expression.

A rat Preprotachykinin-A promoter fragment has been previously identified which supports reporter gene activity in primary cultures of adult dorsal root ganglion neurons. That study demonstrated that two promoter domains which exhibit enhancer activity in these neurons are bound by the same classes of transcription factors. Further, the two domains exhibit similarities with respect to the relationship of bound transcription factors within each domain. This suggests that these domains may function in an identical manner or may act synergistically to regulate gene expression. These domains contain recognition motifs for at least three classes of transcription factors: octamer-binding proteins, Sp1-related proteins and an as yet unidentified but distinct factor. The definition of an octamer-binding protein site within these domains is of interest, as this class of factor has recently been suggested as mediating the effect of nerve growth factor in sensory neurons. Nerve growth factor is a well-characterized regulator of preprotachykinin-A gene expression. Definition of these sites within the promoter allows for the design of rational experiments to address the mechanism of transcriptional regulation of the rat preprotachykinin-A gene.

Interleukin-1 receptor expression by ovine afferent lymph dendritic cells: response to secondary antigen challenge.

Interleukin (IL)-1 is thought to enhance the function of antigen presenting cells of the dendritic cell lineage. To investigate the interaction of IL-1 and dendritic cells recombinant ovine IL-1 alpha and IL-1 beta have been used to determine IL-1 receptor (R) expression on fresh dendritic cells (ALDC) collected from cannulated sheep pseudoafferent lymph ducts, both prior to and in response to localized ovalbumin challenge. Resting ovine ALDC express approximately 510 IL-1R per cell for IL-1 alpha (Kd approximately 30 pM) and approximately 350 IL-1R/cell for IL-1 beta (Kd approximately 160 pM). Saturation binding and in situ analyses show an initial transient but dramatic increase in IL-1 alpha binding to ALDC by 4 h in response to ovalbumin challenge of primed sheep. Maximal IL-1R expression, reaching > or = 21700 IL-1R/cell for IL-1 alpha detected by around 48 h, was followed by a gradual return to resting level by 8 days post challenge. Fewer than 0.5% of resting ALDC expressed IL-1R but at least 5% of ALDC bound IL-1 alpha after ovalbumin challenge. There was no evidence of specific up-regulation of receptors for IL-1 beta on these cells. Fresh ovine alveolar macrophages, used as a positive control for specific IL-1R expression, were found to express approximately 2600 sites/cell for IL-1 alpha (Kd approximately 56 pM) and 16,500 sites/cell for IL-1 beta (Kd approximately 4.6 pM). In view of the differing IL-1 binding characteristics displayed by the receptors on the two cell types, it is postulated that afferent lymph dendritic cells and macrophages are not expressing the same form of IL-1R.

Cloning, expression and characterization of ovine interleukins 1 alpha and beta.

Ovine interleukin 1 alpha (IL-1 alpha) c-DNA, obtained by polymerase chain reaction, has been cloned into pTZ18R and pTZ19R. The resulting DNA sequence shows close homology with the bovine sequence. The derived amino-acid sequence shows conserved motifs similar to those observed in all species studied so far. No signal peptide is seen. Northern blots of RNA from lipopolysaccharide (LPS)-stimulated ovine alveolar macrophages show IL-1 beta m-RNA to be produced earlier than and to be more transient than IL-1 alpha m-RNA. c-DNAs coding for the IL-1 alpha proprotein and IL-1 alpha and IL-1 beta mature proteins have been cloned and expressed in the yeast Ty-VLP system as fusion proteins. The resultant IL-1 protein preparations, cleaved from their fusion partners by the action of activated coagulation Factor Xa, are 80-95% pure and show biological activity in standard thymocyte co-mitogen and cartilage degradation assays for IL-1. Some species specificity is observed in that sheep thymocytes are more responsive to ovine rIL-1 than are mouse thymocytes. The presence of a Factor Xa cleavage site in the IL-1 alpha proprotein suggests that Factor Xa may be involved in the processing of ovine IL-1 alpha to its mature form.

Radioimmunoassay of beta-thromboglobulin. False high values from frozen plasma samples.

Aliquots of plasma samples assayed after storage at -26 degrees C can show a significantly higher level of measured beta-thromboglobulin (beta-TG) than the corresponding unstored aliquots. Blood spun at the recommended speed of 1,800 X g still contains platelets that will release beta-TG on freezing to yield false results--a point that has not been stressed in the literature. Aliquots of blood plasma spun at either 1,800 X g (low speed [LS]) or 20,000 X g (high speed [HS]) were assayed by radioimmunoassay fresh and after storage at 4 degrees C or -26 degrees C. Storage at -26 degrees C increased mean beta-TG values of fresh or 4 degrees C stored LS samples from 0.78 to 1.94 nmol/L (28-70 ng/mL), whereas all HS mean values were +/- 0.51 nmol/L (between 18 and 19 ng/mL). The authors believe that in addition to the accepted precautions regarding blood sampling and handling, it is essential to stress that for accurate measurement of beta-TG in plasma, samples should be centrifuged at HS or, if this is not feasible, that they be assayed as soon as possible without prior freezing.

Effect of nifedipine and mefruside on renal function and platelet function in hypertensive patients.

A study was carried out in 16 patients with moderately severe hypertension to investigate the effects of nifedipine, given alone or combined with a diuretic, on blood pressure and on renal and platelet function. After 4 weeks on placebo, patients were randomized to receive treatment for 6 weeks with either 20 mg, nifedipine twice daily or 25 mg mefruside once daily on a double-blind, double-dummy basis. All patients then received treatment for a further 6 weeks with a combination of the two drugs in the same dosage as before. The results of blood pressure measurements and laboratory investigations during the three phases of the study showed that significantly better blood pressure control was achieved with nifedipine alone than with mefruside alone. Mefruside had an additional hypotensive effect when added to nifedipine. There was no significant change in the renal blood flow or glomerular filtration rate, with a satisfactory control of blood pressure. There was also no detectable change in platelet aggregation with increasing concentrations of ADP and ristocetin. An adaptive mechanism could be responsible for the apparent lack of change compared with single dose studies.

Double-blind randomized trial of the effect of ticlopidine in arteriovenous fistulas for hemodialysis.

The antiplatelet drug ticlopidine was assessed as an agent for improving the patency of Brescia-Cimino arteriovenous fistulas as access for hemodialysis. In a double-blind randomized study over 1 month, two of six fistulas in the ticlopidine group and five of nine in the placebo group failed. A further one placebo and two ticlopidine patients still had functioning fistulas at the time of withdrawal for technical reasons from the trial. Ticlopidine appears, therefore, to enhance the efficacy of Brescia-Cimino fistulas, at least in the short term.