Estrogen receptors in human preadipocytes

Estrogen influences regional adipose tissue distribution and the accompanying cardiovascular disease risk. To elucidate the mechanisms of this link further, we assessed whether human preadipocytes (PAs) expressed estrogen receptors (ERs) and whether there were any regional or gender differences in ER complement. Human PAs expressed the ER alpha gene but not ERP by reverse transcriptase-polymerase chain reaction, possessed ER alpha protein on Western blotting, and displayed specific 17 beta -estradiol (E-2) binding with calculated dissociation constants of 0.78 nM, 0.96 nM, and 1.19 nM and maximal binding capacities of 9.3 fmol/mg, 14.6 fmol/ mg, and 18.2 fmol/mg from three whole cell binding assays. There were no regional differences in ER alpha complement for males or females. There were no gender differences in ER alpha complement for subcutaneous or visceral samples. We conclude that ER alpha but not ERP is present in human PAs. This suggests that the effect of estrogen on adipose tissue deposition has a contribution from the direct effect of estrogen on human PAs via ER alpha.

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57X FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells. (C) 2002 Elsevier Science (USA).

Inducible system in human hepatoma cell lines for hepatitis C virus production

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetra cycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 In to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect naive Huh7 and stable Huh7-human CD81 cells. (C) 2002 Elsevier Science (USA).

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC-tandem mass spectrometry. Whole blood samples (500 mul) were prepared by protein precipitation, followed by C-18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 mug/l (r(2) > 0.996, n = 9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n = 5) at 0.5, 1.2, 20.0, and 75.0 mug/l, was 100.3-105.4% and less than or equal to7.6%, respectively. The assay had a mean relative recovery of 94.8 +/- 3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at -80 degreesC for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze-thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase 11 and III clinical trials. (C) 2002 Elsevier Science B.V. All rights reserved.

Placental growth hormone is not suppressed by oral glucose loading in normal human pregnancy

Placental growth hormone (PGH) progressively replaces pituitary growth hormone in the maternal circulation from mid-gestation onwards in human pregnancy. Our previous investigations have shown that placental growth hormone concentrations correlate well with foetal growth. Despite the apparent correlation between PGH and birthweight, the physiology of its secretion during pregnancy has not been well defined. We investigated the response of maternal serum PCH to oral glucose loading in pregnant women (n = 24) who demonstrated normal glucose tolerance at a mean gestation of 29 weeks. Mean (SEM) fasting PGH concentrations were high (36.9 [6.4] ng/ml). No suppression of PGH was noted at one, two or three hours after a 75 g oral glucose load. Similarly, no changes were noted in growth hormone binding protein or in calculated free PGH over the course of the glucose tolerance test. As expected, insulin concentrations rose sixfold and insulin like growth factor binding protein 1 concentrations fell by 20% with glucose loading. Cot-relation analysis showed maternal weight, BMI, fasting serum glucose serum insulin to be significantly correlated with the babies' birthweight. Our results support the proposition that PGH concentrations in maternal serum are not Suppressed by oral glucose loading in non-diabetic mothers.

The role of conserved CXXXRXG motif in the expression and function of the human norepinephrine transporter

Highly conserved motifs in the monoamine transporters, e.g. the human norepinephrine transporter (hNET) GXXXRXG motif which was the focus of the present study, are likely to be important structural features in determining function. This motif was investigated by mutating the glycines to glutamate (causing loss of function) and alanine, and the arginine to glycine. The effects of hG117A, hR121G and hG123A mutations on function were examined in COS-7 cells and compared to hNET. Substrate K-m values were decreased for hG117A and hG123A, and their K values for inhibition of [3 H]nisoxetine binding were decreased 3-4-fold and 4-6-fold, respectively. Transporter turnover was reduced to 65% of hNET for hG117A and hR121G and to 28% for hG123A, suggesting that substrate translocation is impaired. K values of nisoxetine and desipramine for inhibition of [H-3]norepinephrine uptake were increased by 5-fold for hG117A, with no change for cocaine. The K-i value of cocaine was increased by 3-fold for hG123A, with no change for nisoxetine and desipramine. However, there were no effects of the mutations on the K-d of [H-3]nisoxetine binding or K-i values of desipramine or cocaine for inhibition of [H-3]nisoxetine binding. Hence, glycine residues of the GXXXRXG motif are important determinants of NET expression and function, while the arginine residue does not have a major role. This study also showed that antidepressants and psychostimulants have different NET binding sites and provided the first evidence that different sites on the NET are involved in the binding of inhibitors and their competitive inhibition of substrate uptake. (C) 2002 Elsevier Science B.V. All rights reserved.

Kunjin virus replicon vectors for human immunodeficiency virus vaccine development

We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8(+) T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8(+) T-cell responses. Two immunizations with KUNgag replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of greater than or equal to1:10,000. Immunization with KUNgag replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8(+) T-cell responses, with one immunization of KUNgag VLPs inducing 4.5-fold-more CD8(+) T cells than the number induced after immunization with recombinant vaccinia virus carrying the gag gene (rVVgag). Two immunizations with KUNgag VLPs also provided significant protection against challenge with rVVgag. Importantly, KUN replicon VLP vaccinations induced long-lasting immune responses with CD8(+) T cells able to secrete gamma interferon and to mediate protection 6 to 10 months after immunization. These results illustrate the potential value of the KUN replicon vectors for human immunodeficiency virus vaccine design.

Molecular assays for detection of human metapneumovirus

The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections. We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR). The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe. The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease. These samples were negative for other common microbial causes of respiratory tract disease. We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001. To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform. The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches. We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

Induction of metallothionein in human skin by routine exposure to sunlight: Evidence for a systemic response and enhanced induction at certain body sites

Expression of metallothionein, an antioxidant induced by a variety of stimuli including ultraviolet light, was quantitated by immunohistochemistry in the skin of males aged over 50 who had known short- and long-term exposures to sunlight. Skin punch biopsies were taken from two sites in each subject: the hand in all subjects and a range of other sites matched to patients with a previously excised primary melanoma. Metallothionein expression (strongest in the basal layers of the epidermis and primarily nuclear) was associated with both short- and long-term exposure to sunlight. A plateau of staining intensity was reached after 3 h sun exposure, within the previous 3 d before biopsy. Expression was also elevated in the nonexposed skin sites of subjects who had recent sun exposure, indicating a systemic response to exposure of remote sites. Using the skin of the hand to normalize responses to chronic exposure between individuals, the systemically modulated response to sunlight was significantly greater on the unexposed back than on other sites. The possibility of ultraviolet-induced cytokines selectively modifying the response of skin on a site-specific basis was investigated. The circulating leukocytes, but not lymphocytes, of two individuals exposed to 1 minimal erythema dose whole-body solar-simulated ultraviolet showed increased interleukin-6 mRNA 4 h after exposure. Interleukin-6 was not directly induced in these cell populations 4 h after ultraviolet A or ultraviolet B irradiation ex vivo . Leukocytes may therefore contribute to and amplify the systemic effects of ultraviolet-induced interleukin-6 and metallothionein expression.

(-)-CGP 12177 increases contractile force and hastens relaxation of human myocardial preparations through a propranolol-resistant state of the beta1-adrenoceptor

Two forms of the activated beta(1)-adrenoceptor exist, one that is stabilized by (-)-noradrenaline and is sensitive to blockade by (-)-propranolol and another which is stabilized by partial agonists such as (-)-pindolol and (-)-CGP 12177 but is relatively insensitive to (-)-propranolol. We investigated the effects of stimulation of the propranolol-resistant PI-adrenoceptor in the human heart. Myocardium from non-failing and failing human hearts were set up to contract at 1 Hz. In right atrium from non-ailing hearts in the presence of 200 nM (-)-propranolol, (-)-CGP 12177 caused concentration-dependent increases in contractile force (-logEC(50)[M] 7.3+/-0.1, E-max 23+/-1% relative to maximal (-)-isoprenaline stimulation of beta(1)- and beta(2)-adrenoceptors, n=86 patients), shortening of the time to reach peak force (-logEC(50)[M] 7.4+/-0.1, E-max 37+/-5%, n=61 patients) and shortening of the time to reach 50% relaxation (t(50%), -logEC(50)[M] 7.3+/-0.1, E-max 33+/-2%, n=61 patients). The potency and maxima of the positive inotropic effects were independent of Ser49Gly- and Gly389Arg-beta(1)-adrenoceptor polymorphisms but were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (-logEC(50)[M] 7.7+/-0.1, E-max 68+/-6%, n=6 patients, P

Codon usage bias and A+T content variation in human papillomavirus genomes

We analyzed the codon usage bias of eight open reading frames (ORFs) across up to 79 human papillomavirus (HPV) genotypes from three distinct phylogenetic groups. All eight ORFs across HPV genotypes show a strong codon usage bias, amongst degenerately encoded amino acids, toward 18 codons mainly with T at the 3rd position. For all 18 degenerately encoded amino acids, codon preferences amongst human and animal PV ORFs are significantly different from those averaged across mammalian genes. Across the HPV types, the L2 ORFs show the highest codon usage bias (73.2 +/- 1.6% and the E4 ORFs the lowest (51.1 +/- 0.5%), reflecting as similar bias in codon 3rd position A + T content (L2: 76.1 +/- 4.2%; E4: 58.6 +/- 4.5%). The E4 ORF, uniquely amongst the HPV ORFs, is G + C rich, while the other ORFs are A + T rich. Codon usage bias correlates positively with A + T content at the codon 3rd position in the E2, E6, L1 and L2 ORFs, but negatively in the E4 ORFs. A general conservation of preferred codon usage across human and non-human PV genotypes whether they originate from a same supergroup or not, together with observed difference between the preferred codon usage for HPV ORFs and for genes of the cells they infect, suggests that specific codon usage bias and A + T content variation may somehow increase the replicational fitness of HPVs in mammalian epithelial cells, and have practical implications for gene therapy of HPV infection. (C) 2003 Elsevier B.V. All rights reserved.

Transdermal penetration of vasoconstrictors - Present understanding and assessment of the human epidermal flux and retention of free bases and ion-pairs

Purpose. As reductions in dermal clearance increase the residence time of solutes in the skin and underlying tissues we compared the topical penetration of potentially useful vasoconstrictors (VCs) through human epidermis as both free bases and ion-pairs with salicylic acid (SA). Methods. We determined the in vitro epidermal flux of ephedrine, naphazoline, oxymetazoline, phenylephrine, and xylometazoline applied as saturated solutions in propylene glycol: water (1: 1) and of ephedrine, naphazoline and tetrahydrozoline as 10% solutions of 1: 1 molar ratio ion-pairs with SA in liquid paraffin. Results. As free bases, ephedrine had the highest maximal flux, Jmax = 77.4 +/- 11.7 mug/cm(2)/h, being 4-fold higher than tetrahydrozoline and xylometazoline, 6-fold higher than phenylephrine, 10-fold higher than naphazoline and 100-fold higher than oxymetazoline. Stepwise regression of solute physicochemical properties identified melting point as the most significant predictor of flux. As ion-pairs with SA, ephedrine and naphazoline had similar fluxes (11.5 +/- 2.3 and 12.0 +/- 1.6 mug/cm(2)/h respectively), whereas tetrahydrozoline was approximately 3-fold slower. Corresponding fluxes of SA from the ion-pairs were 18.6 +/- 0.6, 7.8 +/- 0.8 and 1.1 +/- 0.1 respectively. Transdermal transport of VC's is discussed. Conclusions. Epidermal retention of VCs and SA did not correspond to their molar ratio on application and confirmed that following partitioning into the stratum corneum, ion-pairs separate and further penetration is governed by individual solute characteristics.

Do we need a “new” international convention that helps to avoid trafficking in organs? Some “criminal (and civil) law aspects” - Convention on Human Rights and Biomedicine – updated or outdated?

Abstract: § 1 «Do we need a “new” international convention that helps to avoid trafficking in organs? Some criminal (and civil) law aspects”» - «Convention on Human Rights and Biomedicine – updated or outdated?». § 2 Some important connections: on the one hand, between the 1997 Council of Europe Convention on Human Rights and Biomedicine; the 2002 Additional Protocol to the Convention on Human Rights and Biomedicine concerning Transplantation of Organs and Tissues of Human Origin; and, on the other hand, the problem of trafficking in organs, tissues and cells and trafficking in human beings for the purpose of the removal organs. Some connections. § 3 The «international undisputed principle». § 4 Trafficking in organs, tissues and cells; and trafficking in human beings for the purpose of the removal organs. Criminal Law and Civil Law. § 5 Promote organ donation. § 6 The necessity to collect reliable data on both trafficking cases. § 7 The necessity for an internationally agreed definition of trafficking in OTC: Convention on Human Rights and Biomedicine – updated or outdated? § 8 The (inter)national and (il)legal organ («tissue and cell») trade: some cases and some conclusions. § 9 Do we need a new international convention to prevent trafficking in organs, tissues and cells (OTC)? § 10 Of course we need a «new» international convention to prevent trafficking in organs, tissues and cells (OTC). § 11 At the present moment, we do not need a «new» international convention to prevent trafficking in human beings for the purpose of the removal organs. § 12 The Portuguese case. § 13 «Final conclusions.» § Resumo: § 1 «Precisamos de uma "nova" convenção internacional que ajude a evitar o tráfico de órgãos? Alguns aspectos de lei criminal (e civil)» - «Convenção sobre Direitos Humanos e Biomedicina - Actualizada ou desactualizada?». § 2 Algumas conexões importantes: por um lado, entre a Convenção do Conselho da Europa de 1997 sobre Direitos Humanos e Biomedicina; o Protocolo Adicional de 2002 à Convenção sobre os Direitos do Homem e da Biomedicina relativo ao transplante de órgãos e tecidos de origem humana, e, por outro lado, o problema do tráfico de órgãos, tecidos e células e tráfico de seres humanos para fins de remoção dos órgãos. § 3 O «indiscutível princípio internacional». § 4 O Tráfico de órgãos, tecidos e células; e o tráfico de seres humanos para fins de remoção dos órgãos. Direito Penal e Direito Civil. § 5 Promover a doação de órgãos. § 6 A necessidade de colectar dados fidedignos sobre os dois casos de tráfico. § 7 A necessidade de uma definição internacionalmente acordada de tráfico de OTC: Convenção sobre Direitos Humanos e Biomedicina - actualizada ou desactualizada? § 8 A (inter)nacional e (il)legal comercialização de órgãos («de tecidos e de células»): alguns casos e algumas conclusões. § 9 Será que precisamos de uma nova convenção internacional para prevenir o tráfico de órgãos, tecidos e células (OTC)? § 10 É claro que precisamos de uma «nova» convenção internacional para prevenir o tráfico de órgãos, tecidos e células (OTC). § 11 No presente momento, não precisamos de uma «nova» convenção internacional para impedir o tráfico de seres humanos para fins de remoção dos órgãos. § 12 O caso Português. § 13 «As conclusões finais.»