Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL.

The regulatory-targeting subunit (RGL), also called GM) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/RGL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of RGL. To investigate the function of the phosphatase, RGL knockout mice were generated. Animals lacking RGL show no obvious defects. The RGL protein is absent from the skeletal and cardiac muscle of null mutants and present at approximately 50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by approximately 50% in the RGL-deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant RGL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by approximately 90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and RGL null mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that RGL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/RGL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.

The immortalizing and transforming ability of two common human papillomavirus 16 E6 variants with different prevalences in cervical cancer.

Persistent infection with high-risk human papillomaviruses (HPVs), especially type 16 has been undeniably linked to cervical cancer. The Asian-American (AA) variant of HPV16 is more common in the Americas than the prototype in cervical cancer. The different prevalence is based on three amino acid changes within the E6 protein denoted Q14H/H78Y/L83V. To investigate the mechanism(s) behind this observation, both E6 proteins, in the presence of E7, were evaluated for their ability to extend the life span of and transform primary human foreskin keratinocytes (PHFKs). Long-term cell culture studies resulted in death at passage 9 of vector-transduced PHFKs (negative control), but survival of both E6 PHFKs to passage 65 (and beyond). Compared with E6/E7 PHFKs, AA/E7 PHFKs were significantly faster dividing, developed larger cells in monolayer cultures, showed double the epithelial thickness and expressed cytokeratin 10 when grown as organotypic raft cultures. Telomerase activation and p53 inactivation, two hallmarks of immortalization, were not significantly different between the two populations. Both were resistant to anoikis at later passages, but only AA/E7 PHFKs acquired the capacity for in vitro transformation. Proteomic analysis revealed markedly different protein patterns between E6/E7 and AA/E7, particularly with respect to key cellular metabolic enzymes. Our results provide new insights into the reasons underlying the greater prevalence of the AA variant in cervical cancer as evidenced by characteristics associated with higher oncogenic potential.

Genetic validity of RAPD markers at the intra- and inter-specific level in wild Brassica species with n=9.

The sequence homology of co-migrating RAPD markers within a genus, across species, and among populations of a species was investigated. DNA was isolated from ten wild Brassica species with n=9 and the RAPD patterns were established using three random primers. Five RAPD markers which appeared to be characteristic for the n=9 species (genus level), four markers which appeared to be species specific, and one population-specific marker were isolated from agarose gels and hybridized to the RAPD profiles of the ten Brassica species. Two RAPD markers were cloned for comparison with gel-isolated RAPD fragment probes in hybridization experiments. Non-specific and background hybridization, occurring when gel-isolated fragments were used as probes, disappeared when cloned fragments were used. A total of 250 RAPD-marker hybridizations were scored according to visual presence or absence in a gel lane. All except three markers hybridized as expected, resulting in an error rate of 1.2%. The deviating results included a lack of hybridization although a band was visible in the gel, a length polymorphism for one marker, and a dual hybridization signal for two single-band markers.

Relationships of wild Brassica species with chromosome number 2n = 18, based on RFLP studies.

An array of 10 wild Brassica species with chromosome number 2n = 18 represented by 34 populations was analyzed for genome similarity using genomic and cDNA clones. Species studied included B. bourgeaui (Webb) O. Kuntze, B. cretica Lam., B. hilarionis G.E. Post, B. incana Ten., B. insularis Moris, B. macrocarpa (Guss.) Caruel, B. montana Pourret, B. oleracea L., B. rupestris Rafin., and B. villosa Biv. The RFLP data were used to calculate similarities between populations that were subsequently treated in a cluster analysis. Most populations of a species were grouped together and were separate from populations of other species. The previously identified B. incana - B. rupestris - B. villosa complex was verified, and genetic similarity between the species B. montana and B. oleracea was evident. An interesting association between B. insularis and B. macrocarpa was observed. The UPGMA analysis showed that the species tended to cluster according to geographic region: B. cretica and B. hilarionis comprise a cluster that could be called Eastern Mediterranean; B. oleracea, B. bourgeaui, and B. montana define an Atlantic - Western Mediterranean cluster; B. incana, B. rupestris, and B. villosa form an Italian group; and a B. insularis - B. macrocarpa association may be called Central Mediterranean.

Gene structure and expression of the targeting subunit, RGL, of the muscle-specific glycogen-associated type 1 protein phosphatase, PP1G.

The type I phosphatase associated with glycogen, PP1G, plays an important role in glycogen metabolism. PP1G is targeted to glycogen by the R(GL) subunit, which regulates the function of the enzyme. We report the cloning and characterization of the gene as well as the pattern of expression of the R(GL) subunit from mouse. The gene covers more than 37 kb, is composed of four exons and three introns, and codes for a 1089 residue polypeptide with a calculated molecular weight of 121,000. The amino acid sequence has 60% identity with the human and rabbit R(GL). The 5' flanking region of the gene contains a TATA box, c-Myc sites, and a potential cAMP-responsive element. Muscle specific motifs, such as MyoD and MEF-2, were also found. The A-T rich 3'-UTR contained several polyadenylation signals, two associated with poly(A) down-stream consensus motifs. ARE elements, which regulate mRNA stability, were dispersed throughout the 3'-UTR. Northern analysis of poly(A) mRNA from various murine tissues indicates a major transcript of 7.5 kb in skeletal muscle and heart. Western analysis demonstrates that R(GL) protein is present in skeletal and cardiac muscle from mouse, rat, and rabbit but not in L6 myoblasts, L6 myotubes, 3T3 L1 fibroblasts, 3T3 L1 or rat primary adipocytes, confirming that expression of the gene is specific to striated muscle. Analysis of skeletal muscle from rats made diabetic by streptozotocin treatment reveals that the level of R(GL) protein is the same as in control animals, indicating that expression is not regulated by insulin.