Identification of betaIII- and betaIV-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): antibody mapping and cDNA sequencing

Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptation we have further characterized cod tubulins using alpha- and beta-tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain beta-tubulin isotype has been sequenced. In mammals there are five beta-tubulins (betaI, betaII, betaIII, betaIVa and betaIVb) expressed in brain. A cod betaIII-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The betaIII-like tubulin accounted for more than 30% of total brain beta-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated beta(x), which was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was identified as a betaIV-tubulin, and a betaIV-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubulin. Based on the estimated amounts of betaIII- and betaIV-tubulins in cod brain, our results indicate that cod has at least one additional beta-tubulin isotype and that beta-tubulin diversity evolved early during fish evolution. The sequenced cod betaIV-tubulin had four unique amino acid substitutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Optimized conjugation of a fluorescent label to proteins via intein-mediated activation and ligation

Intein-mediated ligation provides a site-specific method for the attachment of molecular probes to proteins. The method is inherently flexible with regard to either the protein sequence or the attached probe, but practical difficulties have limited the widespread use of this valuable labeling system for the attachment of small- to medium-sized molecules. We report herein studies to improve the efficiency and practical application of these reactions, including the assembly of plasmids for the expression of target-intein fusion proteins and the analysis of their reaction with a fluorescent cysteine derivative under a range of conditions. Optimal ligation of the fluorophore to the target protein is critically dependent on the degree of oxidation of the fluorescent cysteine derivative. Efficient ligation has been achieved with freshly prepared fluorescent cysteine derivative under rigorously anaerobic conditions. Similar ligation yields have also been achieved using more practically convenient conditions including anaerobic reaction with addition of thiophenol, or aerobic reaction with the further addition of tricarboxyethylphosphine.     

Pathogen frequency in an age-structured population of<Emphasis Type="Italic"> Plantago lanceolata</Emphasis>

Life-history traits can play important roles in determining the course of ecological species interactions. We explored the consequences of host age on a host-pathogen interaction by quantifying pathogen frequency in an age-structured host population. Our project was motivated by an interest in whether the demographic structure of a host population has consequences for species interactions. In 2 successive years, we planted large cohorts of the perennial herb Plantago lanceolata in its natural environment and observed infection by Fusarium moniliforme, a non-lethal floral fungal pathogen, over 3 years. We documented substantial variation of pathogen frequency across years and between cohorts. Logistic regression revealed that pathogen frequency increased with the number of inflorescences produced and with evidence of prior pathogen presence, whereas it decreased with increasing plant longevity. In addition, interannual variation and an age-year interaction contributed to the observed pathogen frequencies. There was a significant positive effect of age on pathogen frequency overall, but this was not consistent over all ages. Pathogen frequency was higher in 2-year-old plants than in 1-year-olds, suggesting that age-structure can influence the host-pathogen interaction. This pattern did not continue into 3-year-old plants. A possible explanation for this is that selective mortality allows only generally robust plants, and consequently the most resistant plants, to survive to the oldest ages.     

Overexpression of glycogen synthase in mouse muscle results in less branched glycogen

Glycogen, a branched polymer of glucose, serves as an energy reserve in many organisms. The degree of branching likely reflects the balance between the activities of glycogen synthase and branching enzyme. Mice overexpressing constitutively active glycogen synthase in skeletal muscle (GSL30) have elevated muscle glycogen. To test whether excess glycogen synthase activity affected glycogen branching, we examined the glycogen from skeletal muscle of GSL30 mice. The absorption spectrum of muscle glycogen determined in the presence of iodine was shifted to higher wavelengths in the GSL30 animals, consistent with a decrease in the degree of branching. As judged by Western blotting, the levels of glycogenin and the branching enzyme were also elevated. Branching enzyme activity also increased approximately threefold. However, this compared with an increase in glycogen synthase of some 50-fold, so that the increase in branching enzyme in response to overexpression of glycogen synthase was insufficient to synthesize normally branched glycogen.     

Nutrient-regulated protein kinases in budding yeast

The ability of cells to react appropriately to nutritional cues is of fundamental importance, and in budding yeast, a small number of intracellular protein kinases, PKA, Snf1p/AMP-activated kinase, TOR, Gcn2p, and the cyclin-dependent kinase Pho85p have key roles. A recently characterized enzyme, PAS kinase, may be a new member of this group of nutritional transducers.     

GNIP, a novel protein that binds and activates glycogenin, the self-glucosylating initiator of glycogen biosynthesis

Glycogenin is a self-glucosylating protein involved in the initiation of glycogen biosynthesis. Self-glucosylation leads to the formation of an oligosaccharide chain, which, when long enough, supports the action of glycogen synthase to elongate it and form a mature glycogen molecule. To identify possible regulators of glycogenin, the yeast two-hybrid strategy was employed. By using rabbit skeletal muscle glycogenin as a bait, cDNAs encoding three different proteins were isolated from the human skeletal muscle cDNA library. Two of the cDNAs encoded glycogenin and glycogen synthase, respectively, proteins known to be interactors. The third cDNA encoded a polypeptide of unknown function and was designated GNIP (glycogenin interacting protein). Northern blot analysis revealed that GNIP mRNA is highly expressed in skeletal muscle. The gene for GNIP generates at least four isoforms by alternative splicing. The largest isoform GNIP1 contains, from NH(2)- to COOH-terminal, a RING finger, a B box, a putative coiled-coil region, and a B30.2-like motif. The previously identified protein TRIM7 (tripartite motif containing protein 7) is also derived from the GNIP gene and is composed of the RING finger, B box, and coiled-coil regions. The GNIP2 and GNIP3 isoforms consist of the coiled-coil region and B30.2-like domain. Physical interaction between GNIP2 and glycogenin was confirmed by co-immunoprecipitation, and in addition GNIP2 was shown to stimulate glycogenin self-glucosylation 3-4-fold. GNIPs may represent a novel participant in the initiation of glycogen synthesis.     

Effects of targeted overexpression of pleiotrophin on postnatal bone development

Pleiotrophin (PTN) is an extracellular matrix-associated growth/differentiation factor that, in post-natal life, is found mainly in bone and brain. Bone development was investigated in ptn-overexpressing mice between 1 and 30 weeks. In transgenics and controls, PTN (and its receptor syndecan-3) was synthesized by osteoblasts and was present in striated muscle. ptn over-expression enhanced intramembranous bone formation and had multiple effects on long-term bone growth. The pubertal growth spurt did not take place in transgenic mice, in which the growth trajectory was steady and continuous until 25 weeks. By 30 weeks, transgenic and control mice were of the same size, but the calcium content/mg bone was approximately 10% higher in the transgenics. PTN was also localized in growth plate and articular chondrocytes, but only in transgenic mice. In these, synthesis of type I collagen by articular chondrocytes was observed, as well as an encroachment of subchondral bone into the articular cartilage. The results suggest that PTN has multiple roles during in vivo bone formation and remodeling, probably acting as a co-factor or accessory protein that modulates the effects of primary signaling molecules.