The xylA promoter of Bacillus megaterium mediates constitutive gene expression in Escherichia coli

In the present study, we demonstrate that the Escherichia coli–Bacillus megaterium shuttle vector pHIS1522 can be used as a versatile expression vector. Recombinant genes under the control of the xylA promoter are constitutively expressed at a high level in E. coli strains, whereas their expression is strongly induced by the addition of xylose in B. megaterium. The utilization of D ‐xylose is known to be dependent on the xylAB genes in a number of bacteria. For B. megaterium a XylA‐based expression system was established that allows tightly regulated and highly efficient heterologous gene expression. The open reading frame (ORF) of the fluorescent protein turboRFP was cloned under the control of the xylA promoter of B. megaterium in the shuttle vector pHIS1522. Unexpectedly, tRFP expression was not only observed in B. megaterium, but also in E. coli. Based on fluorescence measurements and Western blot analysis, expression was comparable or slightly higher compared with the commonly used pET vectors. Therefore, pHIS1522 can be used as a versatile expression vector in both, B. megaterium and E. coli.     

Lactobacillli expressing llama VHH fragments neutralise <Emphasis Type="Italic">Lactococcus</Emphasis>phages

Background  

Bacteriophages infecting lactic acid bacteria (LAB) are widely acknowledged as the main cause of milk fermentation failures. In this study, we describe the surface-expression as well as the secretion of two functional llama heavy-chain antibody fragments, one binding to the major capsid protein (MCP) and the other to the receptor-binding proteins (RBP) of the lactococcal bacteriophage p2, by lactobacilli in order to neutralise lactococcal phages.     

Exploring gene-environment interactions in Parkinson’s disease

The objective of this study was to explore combined effects of four candidate susceptibility genes and two exposures on Parkinson’s disease (PD) risk; namely, α-synuclein (SNCA) promoter polymorphism REP1, microtubule-associated protein tau (MAPT) H1/H2 haplotypes, apolipoprotein E (APOE) ε2/ε3/ε4 polymorphism, ubiquitin carboxy-terminal esterase L1 (UCHL1) S18Y variant, cigarette smoking and caffeinated coffee consumption. 932 PD patients and 664 control subjects from the NeuroGenetics Research Consortium, with complete data on all six factors, were studied. Uniform protocols were used for diagnosis, recruitment, data collection and genotyping. A logistic regression model which included gene-exposure interactions was applied. Likelihood ratio tests (LRTs) were used for significance testing and Bayesian inference was used to estimate odds ratios (ORs). MAPT (P = 0.007), SNCA REP1 (P = 0.012), smoking (P = 0.001), and coffee (P = 0.011) were associated with PD risk. Two novel interactions were detected: APOE with coffee (P = 0.005), and REP1 with smoking (P = 0.021). While the individual main effects were modest, each yielding OR < 1.6, the effects were cumulative, with some combinations reaching OR = 12.6 (95% CI: 5.9–26.8). This study provides evidence for the long-held notion that PD risk is modulated by cumulative and interactive effects of genes and exposures. Furthermore, the study demonstrates that while interaction studies are useful for exploring risk relationships that might otherwise go undetected, results should be interpreted with caution because of the inherent loss of power due to multiple testing. The novel findings of this study that warrant replication are the evidence for interaction of coffee with APOE, and of smoking with REP1 on PD risk. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Understanding the role of thyroid hormone in Sertoli cell development: a mechanistic hypothesis

More than a decade of research has shown that Sertoli cell proliferation is regulated by thyroid hormone. Neonatal hypothyroidism lengthens the period of Sertoli cell proliferation, leading to increases in Sertoli cell number, testis weight, and daily sperm production (DSP) when euthyroidism is re-established. In contrast, the neonatal Sertoli cell proliferative period is shortened under hyperthyroid conditions, but the mechanism by which thyroid hormone is able to negatively regulate Sertoli cell proliferation has been unclear. Recent progress in the understanding of the cell cycle has provided the opportunity to dissect the molecular targets responsible for thyroid-hormone-mediated effects on Sertoli cell proliferation. In this review, we discuss recent results indicating a critical role for the cyclin-dependent kinase inhibitors (CDKI) p27^Kip1 and p21^Cip1 in establishing Sertoli cell number, testis weight, and DSP, and the ability of thyroid hormone to modulate these CDKIs. Based on these recent results, we propose a working hypothesis for the way in which thyroid hormone regulates the withdrawal of the cell cycle by controlling CDKI degradation. Finally, although Sertoli cells have been shown to have two biologically active thyroid hormone receptor (TR) isoforms, TRα1 and TRβ1, experiments with transgenic mice lacking TRα or TRβ illustrate that only one TR mediates thyroid hormone effects in neonatal Sertoli cells. Although significant gaps in our knowledge still remain, advances have been made toward appreciation of the molecular sequence of events that occur when thyroid hormone stimulates Sertoli cell maturation. We gratefully acknowledge the support of this work by the NIH, USDA, the University of Illinois, the Lalor Foundation, and the Thanis A. Field Endowment at the University of Illinois. D.R. Holsberger was supported by postdoctoral fellowships from the Lalor Foundation and Reproductive Biology Research Training Program (NIH grant T32 HD07028), University of Illinois at Urbana–Champaign.     

Sulfoglucuronyl Neolactoglycolipids in Adult Cerebellum: Specific Absence in Murine Mutants with Purkinje Cell Abnormality

It is shown here that glycolipids of the sulfoglucuronyl neolacto series (SGGLs) are present in the adult rodent cerebellum. SGGLs were not detected in the cerebellar murine mutants lurcher, Purkinje cell degeneration, and staggerer, in which Purkinje cell loss is the primary defect. SGGLs were present, however, in normal amounts in weaver and reeler mutants, in which there is a major and relatively specific loss of granule cells without obvious deficiency in Purkinje cells. In the myelin-deficient quaking mutant, the expression of SGGLs also was nearly normal. The loss of SGGLs in Purkinje cell-deficient mutants was specific, since most of the major lipids were not affected significantly and only the percentage composition of other lipids, such as sulfatides and gangliosides, was altered in the mutants. These and other results strongly suggest that SGGLs and other glycolipids of the paragloboside family are localized specifically in Purkinje cells and their arbors in the adult cerebellum. This is the first demonstration of the localization of a specific glycolipid and its analogs in a specific cell type in the nervous system.     

Chromosomal banding pattern and idiogram of the cotton rat,Sigmodon arizonae (Rodentia,Muridae)

A procedure for obtaining G-bands on chromosomes of mammals is outlined. The procedure was utilized in an investigation of the idiogram and banding pattern of the mitotic chromosomes of the cotton rat, Sigmodon arizonae. The diploid number of this species is 22, and each pair of homologues is easily separated on the basis of size, centromeric position, and banding pattern. The autosomes are represented by four pairs of large submetacentric chromosomes, three pairs of medium to small submetacentric chromosomes, two pairs of large subtelocentric chromosomes and one pair of small acrocentric chromosomes. The X chromosome is acrocentric and averages from 5.42% to 5.46% of the haploid female complement. The Y chromosome is a minute acrocentric and easily separated from the smallest acrocentric autosome. The usefulnes of Sigmodon arizonae as a laboratory animal for cytogenetic studies is substantiated.