A novel cis-acting element, ESP, contributes to high-level endosperm-specific expression in an oat globulin promoter

To examine the genetic controls of endosperm (ES) specificity, several cereal seed storage protein (SSP) promoters were isolated and studied using a transient expression analysis system. An oat globulin promoter (AsGlo1) capable of driving strong ES-specific expression in barley and wheat was identified. Progressive 5' deletions and cis element mutations demonstrated that the mechanism of specificity in the AsGlo1 promoter was distinct from that observed in glutelin and prolamin promoters. A novel interrupted palindromic sequence, ACATGTCAT-CATGT, was required for ES specificity and substantially contributed to expression strength of the AsGlo1 promoter. This sequence was termed the endosperm specificity palindrome (ESP) element. The GCN4 element, which has previously been shown to be required for ES specificity in cereal SSP promoters, had a quantitative role but was not required for tissue specificity. The 960-bp AsGlo1 promoter and a 251-bp deletion containing the ESP element also drove ES-specific expression in stably transformed barley. Reporter gene protein accumulated at very high levels (10% of total soluble protein) in ES tissues of plants transformed with an AsGlo1:GFP construct. Expression strength and tissue specificity were maintained over five transgenic generations. These attributes make the AsGlo1 promoter an ideal promoter for biotechnology applications. In conjunction with previous findings, our data demonstrate that there is more than one genetically distinct mechanism by which ES specificity can be achieved in cereal SSP promoters, and also suggest that there is redundancy between transcriptional and post-transcriptional tissue specificity mechanisms in cereal globulin genes.

Association between chronic fatigue syndrome and the corticosteroid-binding globulin gene ALA SER224 polymorphism

Chronic fatigue syndrome (CFS) is characterized by idiopathic fatigue of greater than 6 months' duration with postexertional exacerbation and many other symptoms. A trend toward relative hypocortisolism is described in CFS. Twin and family studies indicate a substantial genetic etiologic component to CFS. Recently, severe corticosteroid-binding globulin (CBG) gene mutations have been associated with CFS in isolated kindreds. Human leukocyte elastase, an enzyme important in CBG catabolism at inflammatory sites, is reported to be elevated in CFS. We hypothesized that CBG gene polymorphisms may act as a genetic risk factor for CFS. A total of 248 patients with CFS defined by Centers for Disease Control criteria, and 248 controls were recruited. Sequencing and restriction enzyme testing of the CBG gene coding region allowed detection of severe CBG gene mutations and a common exon 3 polymorphism (c.825G --> T, Ala-Ser(224)). Plasma CBG levels were measured in 125 CFS patients and 198 controls by radioimmunoassay. Total and free (calculated and measured) cortisol levels were ascertained in single samples between 8-10 a.m. The age of onset (mid 30s) and gender ratio (2.2:1, female:male) of the patients were similar to those reported in U.S. epidemiologic studies. A trend toward a preponderance of serine(224) homozygosity among the CFS patients was noted, compared with controls (chi(2) = 5.31, P = 0.07). Immunoreactive-CBG (IR-CBG) levels were higher in Serine/Alanine (Ser/Ala) than Ala/Ala subjects and higher again in Ser/Ser subjects, this effect was strongest in controls; Ser/Ser: 46.1 +/- 1.8 (n = 31, P = 0.03) vs. Ser/Ala: 42.4 +/- 1.0 (n = 56, P = 0.05) vs. Ala/Ala: 40.8 +/- 1.7 mug/mL (n = 21). Despite higher CBG levels, there was a nonsignificant trend toward lower total and free plasma cortisol in serine allele positive patients, total cortisol: Ser/Ser: 13.3 +/- 1.4 (n = 34) vs. Ser/Ala: 14.0 +/- 0.7 (n = 66) vs. Ala/Ala: 15.4 +/- 1.0 (n = 23). Homozygosity for the serine allele of the CBG gene may predispose to CFS, perhaps due to an effect on hypothalamic-pituitary-adrenal axis function related to altered CBG-cortisol transport function or immune-cortisol interactions.

Prevalence of HBsAg mutants and impact of hepatitis B infant immunisation in four Pacific Island countries

The prevalence rate of hepatitis B virus (HBV) infection in Pacific Island countries is amongst the highest in the world. Hepatitis B immunisation has been incorporated into national programmes at various times, often with erratic supply and coverage, until a regionally co-ordinated programme, which commenced in 1995 ensured adequate supply. The effectiveness of these programmes was recently evaluated in four countries, Vanuatu and Fiji in Melanesia, Tonga in Polynesia and Kiribati in Micronesia. That evaluation established that the programmes had a substantial beneficial impact in preventing chronic hepatitis B infection [Vaccine 18 (2000) 3059]. Several studies of hepatitis B vaccination programmes in endemic countries have identified the potential significance of surface gene mutants as a cause for failure of immunisation. In the study outlined in this paper, we screened infected children and their mothers for the emergence and prevalence of these variants in specimens collected from the four country evaluation. Although the opportunity for the emergence of HBV vaccine escape mutants in these populations was high due to the presence of a considerable amount of the virus in the population and the selection pressure from vaccine use, there were no a determinant vaccine escape mutants found. This suggests that vaccine escape variants are not an important cause for failure to prevent HBV transmission in this setting. Other HBsAg variants were detected, but their functional significance remains to be determined. The failure to provide satisfactory protection during such immunisation programmes reflects the need for achieving and sustaining high vaccine coverage, improving the timeliness of doses as well as improving 'cold-chain' support, rather than the selection of vaccine-escape mutants of HBV. (C) 2004 Elsevier Ltd. All rights reserved.

Autologous hemopoietic stem cell transplantation in severe rheumatoid arthritis: A report from the EBMT and ABMTR

Objective. Since 1996, autologous hemopoietic stem cell transplantation (HSCT) has been used to treat severe rheumatoid arthritis (RA). To date, published reports have been individual cases or series containing small numbers. This study combined the worldwide experience in a single analysis. Methods. The Autoimmune Disease Databases of the European Group for Blood and Marrow Transplantation (EBMT) and the Autologous Blood and Marrow Transplant Registry (ABMTR) were used to identify patients with RA treated with autologous HSCT. Further information relating to patient and treatment-specific variables was obtained by questionnaire. Results. Seventy-six patients were registered from 15 centers. Seventy-three patients had received autologous HSCT, and in 3 patients hematopoietic stem cells (HSC) were mobilized but not transplanted. Transplanted patients (median age 42 yrs, 74% female, 86% rheumatoid factor positive) had been previously treated with a mean of 5 (range 2-9) disease modifying antirheumatic drugs (DMARD). Significant functional impairment was present, with a median Health Assessment Questionnaire (HAQ) score of 1.4 (range 1.1-2.0) and Steinbrocker score mean 2.39 (SD 0.58). The high dose treatment regimen was cyclophosphamide (CYC) alone in the majority of patients, mostly 200 mg/kg (n = 62). Seven patients received anti-thymocyte globulin (ATG) in addition to CYC, 2 patients busulfan and CYC (BuCYC), and one patient CYC with total body irradiation and ATG. One patient received fludarabine with ATG. Following treatment, one patient received bone marrow but the rest received chemotherapy and/or granulocyte colony-stimulating factor mobilized peripheral blood stem cells. The harvest was unmanipulated in 28 patients, the rest receiving some form of lymphocyte depletion, mostly through CD34+ selection. Median followup was 16 months (range 3-55). Responses were measured using the American College of Rheumatology (ACR) criteria. Forty-nine patients (67%) achieved at least ACR 50% response at some point following transplant. There was a significant reduction in the level of disability measured by the HAQ (p < 0.005). Most patients restarted DMARD within 6 months for persistent or recurrent disease activity, which provided disease control in about half the cases. Response was significantly related to seronegative RA (p = 0.02) but not to duration of disease, number of previous DMARD, presence of HLA-DR4, or removal of lymphocytes from the graft. There was no direct transplant related mortality, although one patient, treated with the BuCYC regimen, died 5 months post-transplant from infection and incidental non-small cell lung cancer. Conclusion. Autologous HSCT is a relatively safe form of salvage treatment in severe, resistant RA. In these open label studies significant responses were achieved in most patients, with over 50% achieving an ACR 50 or more response at 12 months. Although the procedure is not curative, recurrent or persistent disease activity may be subsequently controlled in some patients with DMARD. Clinical trials are necessary to develop this approach inpatients with aggressive disease who have failed conventional treatment including anti-tumor necrosis factor agents.

Plasma steroids and steroid-binding capacity in male semelparous dasyurid marsupials (Phascogale tapoatafa) that survive beyond the breeding season in captivity

The semelparous dasyurids display a unique life history, in that all males die within a few weeks of the completion of the breeding season. Studies of several semelparous species have revealed that the male die-off is stress-related, and accompanied by increased plasma androgen and cortisol levels and decreased corticosteroid binding capacity, resulting in suppression of immune and inflammatory responses. This study examines the endocrine profile of male brush-tailed phascogales (Phascogale tapoatafa) that survive beyond the breeding season in captivity. Plasma cortisol, corticosteroid binding globulin and albumin levels were monitored in both males and females and steroid partitioning calculated. Captive males surviving beyond the breeding season did not show the elevation in plasma cortisol and decrease in corticosteroid binding capacity reported in wild males. Plasma albumin concentrations also remained constant during the sampling period. These data indicate that captive males do not undergo the same stress response described in wild populations. (c) 2006 Elsevier Inc. All rights reserved.