Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal. growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its H-1 chemical shifts suggested that its structure was also very similar to native.

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional H-1/N-15 NMR spectroscopy, and we use H-1/N-15 spin relation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C-4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NE Notably, this face is well removed from the putative DNA-binding face of the NE an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

Socio-economic, migrant and geographic differentials in coronary heart disease occurrence in New South Wales

Objective: This study examines the variation in coronary heart disease (CHD) mortality and acute myocardial infarction (AMI) by socio-economic status (SES), country of birth (COB) and geography (urban/rural) in the total population of New South Wales (Australia) in 1991-95. Method: CHD deaths and AMI are from complete enumerations of deaths and hospital admissions, respectively; and population denominators are from census information. Data are examined separately by sex, and comparisons of SES groups (based on municipalities), COB and region are analysed using Poisson regression, after adjustment for age. Results: The study identified higher risk for AMI admissions and CHD mortality in lower SES populations with significant linear trends, for both sexes, adjusted for age, region and COB. According to the population attributable fractions (PAF), 23-41% of the risk of CHD occurrence is due to SES lower than the highest quartile. The higher age-adjusted risk for CHD occurrence in rural and remote populations for both sexes, compared with urban communities, was lessened by adjustment for COB, and all but abolished when also adjusted for SES. COB analysis indicated significantly lower age-adjusted AMI admissions and CHD mortality compared with the Australian-born, Conclusions: Higher risks for CHD in rural populations compared with the capital city (Sydney) are due, in part, to lower SES, lesser migrant composition. Implications: Strategies for reducing CHD differentials should consider demographic factors and the fundamental need to reduce socio-economic inequalities, as well as targeting appropriate prevention measures.

Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a

Activation of the human complement system of plasma proteins during immunological host defense can result in overproduction of potent proinflammatory peptides such as the anaphylatoxin C5a. Excessive levels of C5a are associated with numerous immunoinflammatory diseases, but there is as yet no clinically available antagonist to regulate the effects of C5a. We now describe a series of small molecules derived from the C-terminus of C5a, some of which are the most potent low-molecular-weight C5a receptor antagonists reported to date for the human polymorphonuclear leukocyte (PMN) C5a receptor. H-1 NMR spectroscopy was used to determine solution structures for two cyclic antagonists and to indicate that antagonism is related to a turn conformation, which can be stabilized in cyclic molecules that are preorganized for receptor binding. While several cyclic derivatives were of similar antagonistic potency, the most potent antagonist was a hexapeptide-derived macrocycle AcF[OPdChaWR] with an IC50 = 20 nM against a maximal concentration of C5a (100 nM) on intact human PMNs. Such potent C5a antagonists may be useful probes to investigate the role of C5a in host defenses and to develop therapeutic agents for the treatment of many currently intractable inflammatory conditions.

Distribution of interleukin-2,-4,-10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus

In the present study. MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with S-35-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta(1) were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (T(H)1) and T(H)2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP. (C) 1999 Elsevier Science Ltd. All rights reserved.

Differentiation of the mononuclear phagocyte system during mouse embryogenesis: The role of transcription factor PU.1

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis, Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms-positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fms proteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms-positive phagocytes at 11.5dpc. PU.1(-/-) embryonic stem cells were able to give rise to macrophagelike cells after cultivation in vitro. The results support previous evidence that yolk sac-derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway. (C) 1999 by The American Society of Hematology.

Who thinks what about capitalism? Class consciousness and attitudes to economic institutions

Attitudes to the fundamental economic institutions of capitalism, private ownership of productive property, markets as arenas for securing economic outcomes, and working class rights to associate and to strike, are key dimensions of class consciousness. This paper investigates how class location shapes these attitudes in combination with other factors like employment sector and trade union membership. Using data from the 1995 National Social Science Survey, the paper finds systematic class variation on attitudes to economic institutions that is consistent with respondents endorsing or rejecting class-specific strategies of interest realisation according to their own class circumstances. On some attitudes, class structural effects are additionally moderated by organisational norms associated with public sector employment and mediated by the impact of trade union membership.

The number of 4-cycles in 2-factorizations of K2n minus a 1-factor

In this paper we give a complete solution to problem of determining the number of 4-cycles in a 2-factorization of K-2n\ 1-factor. (C) 2000 Elsevier Science B.V. All rights reserved.

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: Correlations with fetal growth

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases of noninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFSP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-5 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGK at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P

Insulin-like growth factor-I and transferrin mediate growth and survival of Chinese hamster ovary cells

The aim of this investigation was to elucidate the roles of insulin-like growth factor-I (IGF-I) and transferrin in the survival and proliferation of Chinese hamster ovary (CHO) cells upon withdrawal of serum. For this purpose, we employed DNA analysis and now cytometry to compare CHO cell lines expressing either IGF-I alone or IGF-I and transferrin. The ability of cells to cycle and the occurrence of apoptosis were monitored in these cells in serum-free medium. These results indicate that IGF-I alone is able to maintain the viability of CHO cells for an extended length of time in the absence of serum. Transferrin alone does not promote survival or proliferation. Only in the presence of both IGF-I and transferrin do cells survive and proliferate. Therefore, in attached CHO cultures, IGF-I alone does not stimulate cell proliferation but is a requirement for growth in serum-free medium in cooperation with transferrin. We report on the dual role of IGF-I as a survival factor in CHO cells and its interlocking role with transferrin to stimulate cell growth.

The role of economic evaluation in mental health care

Objective: A consequence of the integration of psychiatry into acute and public health medicine is that psychiatrists are being asked to evaluate their services. There is pressure on mental health-care systems because it is recognized that funds should be directed where they can provide the best health outcomes, and also because there are resource constraints which limit our capacity to meet all demands for health care. This pressure can be responded to by evaluation which demonstrates the effectiveness and efficiency of psychiatric treatment. This paper seeks to remind psychiatrists of the fundamental principles of economic evaluation in the hope that these will enable psychiatrists to understand the methods used in evaluation and to work comfortably with evaluators. Method: The paper reviews the basic principles behind economic evaluation, illustrating these with reference to case studies. It describes: (i) the cost of the burden of illness and treatment, and how these costs are measured; (ii) the measurement of treatment outcomes, both as changes in health status and as resources saved; and (iii) the various types of economic evaluation, including cost-minimization, cost-effectiveness, cost-utility and cost-benefit analysis. Results: The advice in the paper provides psychiatrists with the necessary background to work closely with evaluators. A checklist of the critical questions to be addressed is provided as a guide for those undertaking economic evaluations. Conclusions: If psychiatrists are willing to learn the basic principles of economic evaluation and to apply these, they can respond to the challenges of evaluation.

Variation in mortality rates in Australia: correlation with Indigenous status, remoteness and socio-economic deprivation

Background The aim of this study was to study ecological correlations between age-adjusted all-cause mortality rates in Australian statistical divisions and (1) the proportion of residents that self-identify as Indigenous, (2) remoteness, and (3) socio-economic deprivation. Methods All-cause mortality rates for 57 statistical divisions were calculated and directly standardized to the 1997 Australian population in 5-year age groups using Australian Bureau of Statistics (ABS) data. The proportion of residents who self-identified as Indigenous was obtained from the 1996 Census. Remoteness was measured using ARIA (Accessibility and Remoteness Index for Australia) values. Socioeconomic deprivation was measured using SEIFA (Socio-Economic index for Australia) values from the ABS. Results Age-standardized all-cause mortality varies twofold from 5.7 to 11.3 per 1000 across Australian statistical divisions. Strongest correlation was between Indigenous status and mortality (r = 0.69, p < 0.001). correlation between remoteness and mortality was modest (r = 0.39, p = 0.002) as was correlation between socio-economic deprivation and mortality (r = -0.42, p = 0.001). Excluding the three divisions with the highest mortality, a multiple regression model using the logarithm of the adjusted mortality rate as the dependent variable showed that the partial correlation (and hence proportion of the variance explained) for Indigenous status was 0.03 (9 per cent; p = 0.03), for SEIFA score was -0.17 (3 per cent; p = 0.22); and for remoteness was -0.22 (5 per cent; p = 0.13). Collectively, the three variables studied explain 13 per cent of the variability in mortality. Conclusions Ecological correlation exists between all-cause mortality, Indigenous status, remoteness and disadvantage across Australia. The strongest correlation is with indigenous status, and correlation with all three characteristics is weak when the three statistical divisions with the highest mortality rates are excluded. intervention targeted at these three statistical divisions could reduce much of the variability in mortality in Australia.