In a mucB (algN) genetic background, insertion of an ⍀ element ϳ200 bp downstream of glpD, encoding sn-glycerol-3-phosphate dehydrogenase from Pseudomonas aeruginosa, had an adverse effect on alginate biosynthesis from various carbon sources. The insertion inactivated glpM, a gene encoding a 12,040-M r hydrophobic protein containing 109 amino acids. This protein, which was expressed in a T7 RNA polymerase expression system, appears to be a cytoplasmic membrane protein.Mucoid strains of the opportunistic pathogen Pseudomonas aeruginosa isolated from cystic fibrosis patients with chronic pulmonary infections secrete copious amounts of the extracellular polysaccharide alginate (for comprehensive reviews, see references 16 and 17). Whereas over the past few years significant progress has been made toward (i) understanding the complex regulation of the expression of the alginate biosynthetic genes and (ii) characterization of the gene products involved in the biosynthetic process (16,17), little is known about the origin(s) of the carbon moieties found in the alginate molecule. The desire to understand these origins is also driven by our lack of understanding of the availability of carbon in the cystic fibrosis lung, the environment in which mucoid derivatives of P. aeruginosa are most often found and in which they afflict the most damage. Of particular interest to our studies are the previously reported findings that triose phosphates are obligate intermediates in the biosynthesis of alginate (1) and that fructose 1,6-bisphosphate aldolase is essential for this to occur (2). We are therefore focussing our efforts on one peripheral carbon metabolic pathway which is capable of directly providing these triose phosphate intermediates (2), namely the glycerol metabolic pathway.Although some of the basic events involved in glycerol metabolism were studied some time ago (4, 18, 29, 31), we have only recently begun to unravel at the molecular level the pathway involved in glycerol metabolism in P. aeruginosa. As a first step in elucidating the molecular organization and mode(s) of regulation of the glp genes of P. aeruginosa, we have recently cloned the glpD gene, encoding sn-glycerol-3-phosphate dehydrogenase, a key enzyme of the glycerol metabolic pathway (29), and subsequently the genes encoding the glycerol transporter (glpF), glycerol kinase (glpK), and the regulatory gene (glpR) (23) (Fig. 1A). In this communication, we report the observation that insertions in the glpD region have an adverse effect on alginate biosynthesis and that this may be due to inactivation of a closely linked gene, glpM.Insertions in the glpD region have an adverse effect on alginate biosynthesis. Construction of insertion mutants in the glpD region of wild-type strain PAO1 (8) was done by a previously described sacB-based gene replacement procedure (26). The glpD mutant strain PAO151 was obtained by insertion of the tetracycline resistance (Tc r )-encoding ⍀ element from pHP45⍀Tc (5) into the single NotI site (Fig. 1A) within glpD on pEB22⌬E1...

Key regulators of the Wnt signalling, DVL1, DVL2 and DVL3, in astrocytomas of different malignancy grades were investigated. Markers for DVL1,DVL2 and DVL3 were used to detect microsatellite instability (MSI) and gross deletions (LOH), while immunohistochemistry and immunoreactivity score were used to determine the signal strengths of the three DVL proteins and transcription factors of the pathway, TCF1 and LEF1. Our findings demonstrated that MSI at all three DVL loci was constantly found across tumour grades with the highest number in grade II (P = 0.008). Collectively, LOHs were more frequent in high‐grade tumours than in low grade ones. LOHs of DVL3 gene were significantly associated with grade IV tumours (P = 0.007). The results on protein expressions indicated that high‐grade tumours expressed less DVL1 protein as compared with low grade ones. A significant negative correlation was established between DVL1 expression and malignancy grades (P < 0.001). The expression of DVL2 protein was found similar across grades, while DVL3 expression significantly increased with malignancy grades (P < 0.001). The signal strengths of expressed DVL1 and DVL3 were negatively correlated (P = 0.002). However, TCF1 and LEF1 were both significantly upregulated and increasing with astrocytoma grades (P = 0.001). A positive correlation was established between DVL3 and both TCF1 (P = 0.020) and LEF1 (P = 0.006) suggesting their joint involvement in malignant progression. Our findings suggest that DVL1 and DVL2 may be involved during early stages of the disease, while DVL3 may have a role in later phases and together with TCF1 and LEF1 promotes the activation of Wnt signalling.