Plus and minus sexual agglutinins from Chlamydomonas reinhardtii

Gametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins. Both agglutinin proteins are organized into three distinct domains: a head, a shaft in a polyproline II configuration, and an N-terminal domain. The plus and minus heads are related in overall organization but poorly conserved in sequence except for two regions of predicted hydrophobic alpha-helix. The shafts contain numerous repeats of the PPSPX motif previously identified in Gp1, a cell wall HRGP. We propose that the head domains engage in autolectin associations with the distal termini of their own shafts and suggest ways that adhesion may involve head-head interactions, exolectin interactions between the heads and shafts of opposite type, and antiparallel shaft-shaft interactions mediated by carbohydrates displayed in polyproline II configurations.     

Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions

The membrane topology of connexin32, a principal polypeptide of gap junctions in diverse cell types, has been studied in rat and mouse hepatocyte gap junctions using site-specific antisera raised against synthetic oligopeptides corresponding to amino acid sequences deduced from cDNA clones. Based on published hydropathicity maps and identified protease-sensitive cleavage sites, oligopeptides were synthesized corresponding to two hydrophilic domains of connexin32, one predicted to face the cytoplasm, the other predicted to be directed extracellularly. Antisera were raised to keyhole limpet hemocyanin conjugates of the oligopeptides and used to map the distribution of their antigens using indirect immunocytochemistry on isolated gap junctions. The results directly demonstrated the cytoplasmic orientation of an antigen contained within amino acids 98-124 of the connexin32 sequence. The extracellular space in intact, isolated gap junctions is too small to permit binding of antibody molecules, necessitating the experimental separation of the junctional membranes to expose their extracellular surfaces using a urea/alkali procedure. While an antigen contained within amino acids 164-189 was visualized on the extracellular surfaces of some of the separated junctional membranes, variability in the observations and in the splitting procedure left ambiguities concerning the biological relevance of the observations after the denaturing conditions necessary to separate the junctional membranes. Using a different approach, however, the antigen could be exposed in intact liver using a hypertonic disaccharide junction-splitting procedure. The period of time of antigen exposure at the cell surface appears to peak at 30 s and disappear by 2-4 min. Taken together, these data demonstrate the extracellular orientation of an antigen contained within amino acids 164-189, which may be involved in cell-cell interaction within the gap junction.     

Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Fracture prevention in COPD patients; a clinical 5-step approach

Although osteoporosis and its related fractures are common in patients with COPD, patients at high risk of fracture are poorly identified, and consequently, undertreated. Since there are no fracture prevention guidelines available that focus on COPD patients, we developed a clinical approach to improve the identification and treatment of COPD patients at high risk of fracture. We organised a round-table discussion with 8 clinical experts in the field of COPD and fracture prevention in the Netherlands in December 2013. The clinical experts presented a review of the literature on COPD, osteoporosis and fracture prevention. Based on the Dutch fracture prevention guideline, they developed a 5-step clinical approach for fracture prevention in COPD. Thereby, they took into account both classical risk factors for fracture (low body mass index, older age, personal and family history of fracture, immobility, smoking, alcohol intake, use of glucocorticoids and increased fall risk) and COPD-specific risk factors for fracture (severe airflow obstruction, pulmonary exacerbations and oxygen therapy). Severe COPD (defined as postbronchodilator FEV₁ < 50% predicted) was added as COPD-specific risk factor to the list of classical risk factors for fracture. The 5-step clinical approach starts with case finding using clinical risk factors, followed by risk evaluation (dual energy X-ray absorptiometry and imaging of the spine), differential diagnosis, treatment and follow-up. This systematic clinical approach, which is evidence-based and easy-to-use in daily practice by pulmonologists, should contribute to optimise fracture prevention in COPD patients at high risk of fracture.     

Genetic structure of the mating-type locus of Chlamydomonas reinhardtii.

Portions of the cloned mating-type (MT) loci (mt(+) and mt(-)) of Chlamydomonas reinhardtii, defined as the approximately 1-Mb domains of linkage group VI that are under recombinational suppression, were subjected to Northern analysis to elucidate their coding capacity. The four central rearranged segments of the loci were found to contain both housekeeping genes (expressed during several life-cycle stages) and mating-related genes, while the sequences unique to mt(+) or mt(-) carried genes expressed only in the gametic or zygotic phases of the life cycle. One of these genes, Mtd1, is a candidate participant in gametic cell fusion; two others, Mta1 and Ezy2, are candidate participants in the uniparental inheritance of chloroplast DNA. The identified housekeeping genes include Pdk, encoding pyruvate dehydrogenase kinase, and GdcH, encoding glycine decarboxylase complex subunit H. Unusual genetic configurations include three genes whose sequences overlap, one gene that has inserted into the coding region of another, several genes that have been inactivated by rearrangements in the region, and genes that have undergone tandem duplication. This report extends our original conclusion that the MT locus has incurred high levels of mutational change.