Expression of growth factors in Dictyostelium discoideum

Growth factors and their binding proteins are important proteins regulating mammalian cell proliferation and differentiation so there is considerable interest in producing them as recombinant proteins, especially in hosts that do not already produce a complex mixture of growth factors. Many growth factors require posttranslational modifications making them unsuitable for production in Escherichia coli or other prokaryotes. Since several expression vector systems have been recently developed for foreign protein production in the cellular slime mould, Dictyostelium discoideum, we attempted to use two of these systems to express human insulin-like growth factor binding protein 6 (hIGFBP6) and bovine beta-cellulin (bBTC) as secreted proteins. Although both proteins were successfully produced in stably transformed amoebae, no secretion was detected in spite of several attempts to facilitate this occurring.

Estradiol-regulated expression of the immunophilins cyclophilin 40 and FKBP52 in MCF-7 breast cancer cells

The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with the unactivated estrogen receptor in mutually exclusive heterocomplexes and may differentially modulate receptor activity, We have recently shown that CyP40 and FKBP52 mRNA's are differentially elevated in breast carcinomas compared with normal breast tissue. Other studies suggest that such alterations ill the ratio of immunophilins might potentially influence steroid receptor function. Studies were therefore initiated to investigate the influence of estradiol on CyP40 and FKBP52 expression in MCF-7 breast cancer cells. Over a 24-h-treatment period with estradiol, CyP40 and FKBP52 mRNA expression was increased approximately five- and 14-fold, respectively. The corresponding protein levels were also elevated in comparison to controls. The antiestrogen, ICI 182,780, was an antagonist for CyP40 and FKBP52 mRNA induction. Cycloheximide treatment did not inhibit this increased immunophilin expression, suggesting that estradiol-mediated activation is independent off de novo protein synthesis. Treatment of MCF-7 cells with estradiol resulted in an increased half-life of both CyP40 and FKBP52 mRNA, as determined by actinomycin D studies. These results suggest that estradiol regulates CyP40 and FKBP52 mRNA expression through both transcriptional and posttranscriptional mechanisms. (C) 2001 Academic Press.

MHC class II expression is regulated in dendritic cells independently of invariant chain degradation

We have investigated the mechanisms that control MHC class II (MHC II) expression in immature and activated dendritic cells (DC) grown from spleen and bone marrow precursors. Degradation of the MHC II chaperone invariant chain (li), acquisition of peptide cargo by MHC II, and delivery of MHC II-peptide complexes to the cell surface proceeded similarly in both immature and activated DC. However, immature DC reendocytosed and then degraded the MHC II-peptide complexes much faster than the activated DC. MHC II expression in DC is therefore not controlled by the activity of the protease(s) that degrade Ii, but by the rate of endocytosis of peptide-loaded MHC II. Late after activation, DC downregulated MHC II synthesis both in vitro and in vivo.

Social structures, genetic structures and dispersal strategies in Australian rabbit (Oryctolagus cuniculus) populations

In this study, the pattern of movement of young male and female rabbits and the genetic structures present in adult male and female populations in four habitats was examined. The level of philopatry in young animals was found to vary between 18-90% for males and 32-95% for females in different populations. It was skewed, with more males dispersing than females in some but not all populations. Analysis of allozyme data using spatial autocorrelation showed that adult females from the same social group, unlike males, were significantly related in four of the five populations studied. Changes in genetic structure and rate of dispersal were measured before and during the recovery of a population that was artificially reduced in size. There were changes in the rate and distance of dispersal with density and sex. Subadults of both sexes moved further in the first year post crash (low density) than in the following years. While the level of dispersal for females was lower than that of the males for the first 3 years, thereafter (high density) both sexes showed similar, low levels of dispersal (20%). The density at which young animals switch behaviour between dispersal and philopatry differed for males and females. The level of genetic structuring in adult females was high in the precrash population, reduced in the first year post crash and undetectable in the second year. Dispersal behaviour of rabbits both affects the genetic structure of the population and changes with conditions. Over a wide range of levels of philopatry, genetic structuring is present in the adult female, but not the male population. Consequently, though genetic structuring is present, it does not lead to inbreeding. More long-distance movements are found in low-density populations, even though vacant warrens are available near birth warrens. The distances moved decreased as density increased. Calculation of the effective population size (N-e) shows that changes in dispersal distance offset changes in density, so that N-e remains constant.

Using biplots to interpret gene expression patterns in plants

Sum: Plant biologists in fields of ecology, evolution, genetics and breeding frequently use multivariate methods. This paper illustrates Principal Component Analysis (PCA) and Gabriel's biplot as applied to microarray expression data from plant pathology experiments. Availability: An example program in the publicly distributed statistical language R is available from the web site (www.tpp.uq.edu.au) and by e-mail from the contact. Contact: scott.chapman@csiro.au.

Semiochemicals and social signaling in the wild European rabbit in Australia: I. Scent profiles of chin gland secretion from the field

The European rabbit (Oryctolagus cuniculus) uses the secretion of the chin gland in the maintenance of social status. Previous work has concentrated on secretion collected directly from the animal. In this study, the analysis was conducted by collecting scent marks made by free-ranging animals. Scent marks were found to be concentrated at the center of the area controlled by a social group, and at the boundaries between two adjacent social groups. Only the mark from dominant animals could be identified. Marks were also collected from the skin of rabbits, where they had been placed by the dominant individual. The mark found on the head of a subordinate animal may, in the future, be used to identify the dominant animal of the social group, who placed the mark.

Expression of RANTES and CCR1 in oral lichen planus and association with mast cell migration

Background: T lymphocytes and mast cells infiltrate the lamina propria in oral lichen planus (OLP). Chemokines and their receptors are involved in T cell and mast cell migration and accumulation during the inflammatory process. Methods: In the present study, we investigated the role of RANTES and its receptors in OLP using immunohistochemistry, RT-PCR and an in vitro chemotaxis assay. Results: RANTES and CCR1 were expressed on T cells and mast cells in OLP, while OLP lesional T cell supernatants stimulated CCR1 mRNA expression in a human leukemia mast cell line (HMC-1). TNF-alpha stimulated CCR1, CCR4 and CCR5 mRNA expression in the same cell line. OLP lesional T cell supernatants stimulated HMC-1 migration, which was partly inhibited by anti-RANTES antibody. Conclusions: The present study shows, for the first time, the distribution of RANTES and CCR1 in OLR It is hypothesized that RANTES and CCR1 may play important roles in mast cell trafficking and related events in OLP.

Sequence analysis and expression of a virus-like particle protein,VLP2, from the parasitic wasp Venturia canescens

Endoparasitoid wasps produce maternal protein secretions, which are transported into the body of insect hosts at oviposition to regulate host physiology for successful development of their offspring. Venturia canescens calyx fluid contains so-called virus-like particles (VLPs) that are essential for immune evasion of the developing parasitoid inside the host. VLPs consist of four major proteins. In this paper, we describe the isolation and molecular cloning of a gene (vlp2) that is a constituent of VLPs and discuss its possible role in VLP structure and function.

Myb-binding protein 1a augments AhR-dependent gene expression

We have studied the mechanism by which an acidic domain (amino acids 515-583) of the aromatic hydrocarbon receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein la, whereas a mutant domain (F542A, 1569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein la into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein la is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein la associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein la to increase AhR-dependent gene expression.

Variable expression of mental retardation, autism, seizures, and dystonic hand movements in two families with an identical ARX gene mutation

Two families, originally diagnosed as having nonsyndromic X-linked mental retardation (NSXLMR), were reviewed when it was shown that they had a 24-bp duplication (428-45 1dup(24bp)) in the ARX gene [Stromme et al., 2002: Nat Genet 30:441-445]. This same duplication had also been found in three other families: one with X-linked infantile spasms and hypsarrhythmia (X-linked West syndrome, MIM 308350) and two with XLMR and dystonic movements of the hands (Partington syndrome, MIM 309510). On review, manifestations of both West and Partington syndromes were found in some individuals from both families. In addition, it was found that one individual had autism and two had autistic behavior, one of whom had epilepsy. The degree of mental retardation ranged from mild to severe. A GCG trinucleotide expansion (GCG)10+7 and a deletion of 1,517 by in the ARX gene have also been found in association with the West syndrome, and a missense mutation (1058C >T) in a family with a newly recognized form of myoclonic epilepsy, severe mental retardation, and spastic paraplegia [Scheffer et al., 2002: Neurology, in press]. Evidently all these disorders are expressions of mutations in the same gene. It remains to be seen what proportions of patients with infantile spasms, focal dystonia, autism, epilepsy, and nonsyndromic mental retardation are accounted for by mutations in the ARX gene. (C) 2002 Wiley-Liss, Inc.

Outcome research in psychodynamic psychotherapy: Psychological, social and neuropsychological factors

While many studies have demonstrated positive outcomes from psychotherapy when it is practiced in a controlled research environment with carefully selected (or excluded) patient groups and rigid manualised therapy sessions there is a paucity of research regarding effective outcomes from psychotherapy as it is practiced in actual clinical conditions. The aim of this series of studies was to investigate outcomes, using an effectiveness approach, from psychodynamic psychotherapy as it is practiced by private psychiatrists. Three studies were planned. The aim of Study 1 was to provide standardized baseline measures on the following dimensions • Personal Demographic Information (PDI), • Target Symptoms and Disorders (TSD) including a neuropsychological profile • Inter and Intra Personal (IIP) factors, and, • General Functioning and Quality of Life (GFQoL) factors. Study 2 aimed to examine changes in patient characteristics during the course of treatment. Thus, baseline assessments were repeated at sixmonthly intervals to determine if therapy had been effective for individual patients. A third study was planned to assess the extent to which the results of significant outcome predictors could be replicated in different patient samples. Twenty-nine psychiatrists consented to refer patients with 20 patients having completed pre therapy assessments and six and 18-month follow-up questionnaires. The presentation of this research will focus on the interesting research methodology utilized, patient demographic characteristics and on the patient changes occurring over time on the dimensions of Defence Style (DSQ), Quality of Life (WHOQOL- Bref) and the severity of depression (BDI). The patient sample included 10 male and 10 female patients, whose ages ranged from 19 years to 66 years (mean = 43 years). While seven of the patients did not meet SCID-IV criteria for a current DSM-IV Axis 1 disorder, six patients met criteria for a current mood disorder, three for panic disorder, one patient each for PTSD, alcohol abuse and dependence, and 2 patients met current criteria for multiple Axis 1 disorders. The research is ongoing.