Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman(TM) RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P < 0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P < 0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the-codon:reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more Widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin, secretion. (C) 2003 Elsevier Science B.V. All rights reserved.

Application of a species-specific PCR-RFLP to identify Ancylostoma eggs directly from canine faeces

Apart from their veterinary importance, the hookworms Ancylostoma caninum, Ancylostoma braziliense and Ancylostoma caninum are also capable of causing zoonotic disease in humans. A highly sensitive and species-specific PCR-RFLP technique was utilised to detect and differentiate the various canine Ancylostoma spp directly from eggs in faeces. This technique was utilised to screen 101 canine faecal samples from parasite endemic tea growing communities in Assam, India, as part as an ongoing epidemiological investigation into canine parasitic zoonoses. The prevalence of hookworms in dogs was found to be 98% using a combination of PCR and conventional microscopy. Overall, 36% of dogs were found positive for single hookworm infections with A. caninum, 24% positive for single infections with A. braziliense and 38% had mixed infections with both A. caninum and A. braziliense. No dogs were found positive for A. ceylanicum in the community under study. The high prevalence of A. caninum and A. braziliense in dogs in this community may account for the high incidence of cutaneous larva migrans (CLM) observed among the human population residing at the tea estates. The PCR-RFLP technique described herein allows epidemiological screening of canine hookworms to be conducted rapidly, with ease and accuracy, and has the potential to be applied to a number of different clinical, pharmacological and epidemiological situations. (C) 2004 Elsevier B.V. All rights reserved.

A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS-MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues, The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-mul amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C-18 (150X4.6 mm, 5 mum) column at 35 degreesC. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored. (C) 2001 Elsevier Science B.V. All rights reserved.

Low level expression of human papillomavirus type 16 (HPV16) E6 in squamous epithelium does not elicit E6 specific B- or T-helper immunological responses, or influence the outcome of immunisation with E6 protein

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically naive to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of EB-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised Eb-transgenic mice that have not developed inflammatory skin disease remain immunologically naive to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines. (C) 2001 Elsevier Science B.V. All rights reserved.

The rights to a deceased's body and body parts : in the matter of Gray (2000) QSC 390 (12/10/2000).

Decision In the Matter of Gray highlights complications that advancing medical technology causes to the law - case concerns the issue of removal of semen from a deceased man - how the courts deal with matters concerning medical technology in the absence of specific legislation or established case law - legal and moral questions raised by the case.

Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine. (C) 2003 Elsevier Science Ltd. All rights reserved.

Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and in a subset of tissue macrophages and dendritic cells. It is expressed at lower levels in the parenchymal cells of the liver, glomerular mesangial cells of the kidney and pancreatic acinar cells. We have identified novel TRAP mRNAs that differ in their 5-untranslated region (5'-UTR) sequence, but align with the known murine TRAP mRNA from the first base of Exon 2. The novel 5'-UTRs represent alternative first exons located upstream of the known 5'-UTR. A similar genomic structure exists for the human TRAP gene with partial conservation of the exon and promoter sequences. Expression of the most distal 5'-UTR (Exon 1A) is restricted to adult bone and spleen tissue. Exon 1B is expressed primarily in tissues containing TRAP-positive nonhaematopoietic cells. The known TRAP 5'-UTR (Exon 1) is expressed in tissues characteristic of myeloid cell expression. In addition the Exon 1C promoter sequence is shown to comprise distinct transcription start regions, with an osteoclast-specific transcription initiation site identified downstream of a TATA-like element. Macrophages are shown to initiate transcription of the Exon 1C transcript from a purine-rich region located upstream of the osteoclast-specific transcription start point. The distinct expression patterns for each of the TRAP 5'-UTRs suggest that TRAP mRNA expression is regulated by the use of four alternative tissue- and cell-restricted promoters. (C) 2003 Elsevier Science B.V. All rights reserved.

Intra-specific variation for host plant use in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae): Implications for management

The polyphagous moth Helicoverpa armigera (Hubner) is one of the world's most important agricultural pests. A number of existing approaches and future designs for management of H. armigera rely on the assumption that moths do not exhibit either genetically and/or non-genetically based variation for host plant utilization. We review recent empirical evidence demonstrating that both these forms of variation influence host plant use in this moth. The significance of this variation in H. armigera in relation to current and future pest management strategies is examined. We provide recommendations on future research needs and directions for sustainable management of H. armigera, under a framework that includes consideration of intra.-specific variation for host use relevant in this and other similar pest species. (C) 2004 Elsevier Ltd. All rights reserved.

Neck flexor muscle fatigue is side specific in patients with unilateral neck pain

Despite the evidence of greater fatigability of the cervical flexor muscles in neck pain patients, the effect of unilaterality of neck pain on muscle fatigue has not been investigated. This study compared myoelectric manifestations of sternocleidomastoid (SCM) and anterior scalene (AS) muscle fatigue between the painful and non-painful sides in patients with chronic unilateral neck pain. Myoelectric signals were recorded from the sternal head of SCM and the AS muscles bilaterally during sub-maximal isometric cervical flexion contractions at 25% and 50% of the maximum voluntary contraction (MVC). The time course of the mean power frequency, average rectified value and conduction velocity of the electromyographic signals were calculated to quantify myoelectric manifestations of muscle fatigue. Results revealed greater estimates of the initial value and slope of the mean frequency for both the SCM and AS muscles on the side of the patient's neck pain at 25% and 50% of MVC. These results indicate greater myoelectric manifestations of muscle fatigue of the superficial cervical flexor muscles ipsilateral to the side of pain. This suggests a specificity of the effect of pain on muscle function and hence the need for specificity of therapeutic exercise in the management of neck pain patients. (C) 2003 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. All rights reserved.

The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here. we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20,46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 198 1, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets. (C) 2003 Elsevier B.V. All rights reserved.

Ergot of rye - the first specific for migraine

Over the past decade the various triptan derivatives have been accepted as the most effective available agents for relieving migraine attacks. Prior to that, for a period of half a century, ergotamine was the only 'specific' available for this purpose. In 1918, Stoll had isolated it from the various alkaloids present in extracts of the sclerotia of the fungus Claviceps purpurea (ergot), which grow on rye and, to a lesser extent, on other grasses. By 1925 ergotamine was beginning to be used to treat migraine attacks. However, as ergotamine was present in extracts of ergot, which had been used to treat migraine first, In Italy in 1862, and then by Edward Woakes (11868) in England, and after him by Albert Eulenburg in Germany (1883), the drug had actually come into unrecognised use for the disorder more than half a century before ergotamine itself was known to exist. Unfortunately, because of ergotamine's chemical and pharmacokinetic properties, extracts of ergot of rye were incapable of producing consistent therapeutic results, so that general acceptance that the first specific substance for migraine treatment existed had to wait until pure ergotamine was available for administration. (C) 2003 Elsevier Ltd. All rights reserved.

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX (TM) adjuvant in women with cervical intraepithelial neoplasia

Purpose: Persistent infection of cervical epithelium with high risk human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV 16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX(TM) adjuvant (HPV16 Immunotherapeutic) for patients with CIN. Experimental design: Patients with CIN (n = 3 1) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy. Results: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients. Conclusions : The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX(TM) adjuvant is safe and induces vaccine antigen specific cell mediated immunity. (C) 2004 Elsevier Ltd. All rights reserved.

Modulation of proliferation-specific and differentiation-specific markers in human keratinocytes by SMAD7

We examined the potential role of SMAD7 in human epidermal keratinocyte differentiation. Overexpression of SMAD7 inhibited the activity of the proliferation-specific promoters for the keratin 14 and cdc2 genes and reduced the expression of the mRNA for the proliferation-specific genes cdc2 and E2F1. The ability of SMAD7 to suppress cdc2 promoter activity was lost in transformed keratinocyte cell lines and was mediated by a domain(s) located between aa 195-395 of SMAD7. This domain lies outside the domain required to inhibit TGFbeta1 signaling, suggesting that this activity is mediated by a novel functional domain(s). Examination of AP1, NFkappaB, serum response element, Gli, wnt, and E2F responsive reporters indicated that SMAD7 significantly suppressed the E2F responsive reporter and modestly increased AP1 activity in proliferating keratinocytes. These data Suggest that SMAD7 may have a role in TGFbeta-independent signaling events in proliferating/undifferentiated keratinocytes. The effects of SMAD7 in differentiated keratinocytes indicated a more traditional role for SMAD7 as an inhibitor of TGFbeta action. SMAD7 was unable to initiate the expression of differentiation markers but was able to superinduce/derepress differentiation-specific markers and genes in differentiated keratinocytes. This latter role is consistent with the ability of SMAD7 to inhibit TGFbeta-mediated suppression of keratinocyte differentiation and suggest that the opposing actions of SMAD7 and TGFbeta may serve to modulate squamous differentiation. (C) 2004 Elsevier Inc. All rights reserved.

Performance of the Norwegian version of AUSCAN - a disease-specific measure of hand osteoarthritis

Objective: To examine the performance of the Norwegian version of the AUSCAN Index as a disease-specific health status measure in patients with hand osteoarthritis (OA). Methods: One hundred and ninety-nine patients with clinical hand OA (mean (SD) age 61.7 (5.7) years, 18 (9%) males) underwent a comprehensive examination including joint status, examination of grip strength and completion of several self-reported health status questionnaires. The Australian/Canadian OA hand index (AUSCAN) captures three different dimensions of hand OA: pain (5 items), stiffness (1 item), and difficulties with daily activities (9 items). Our pre-study hypothesis was to identify AUSCAN as a specific hand measure with strong correlations to hand measures and lower correlations to other general measures of health. Results: Patient completion of the AUSCAN Index was similar or better than other measures. The internal consistency of the AUSCAN was excellent. The pain and physical dimension of AUSCAN correlated substantially to, each other and moderately to the stiffness scale. The AUSCAN physical scale correlated moderately to substantially to other measures, the highest correlation being seen with the Arthritis Impact Measurement Scale (AIMS) 2 hand and finger function scale (r= 0.73). The standardised differences between patients with and without radiographic abnormalities were numerically larger for the AUSCAN pain and physical scales than for other measures. Conclusion: The Norwegian version of the AUSCAN has an acceptable clinimetric performance and is a suitable tool for assessment of hand OA. (C) 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1 alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1 alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1 alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1 alpha-dependent mechanism. (c) 2005 Elsevier Inc. All rights reserved.