Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes

This report examines in detail the metabolism of the cyclin protein B1 during meiotic maturation and following the activation of mature mouse oocytes using immunoprecipitation of the radiolabelled protein. The net synthesis of cyclin B increases progressively during meiotic maturation, reaching its maximum levels at least 1 h before oocytes exit into metaphase of meiosis II (MII). This increase correlates with the rise in cdc2 kinase activity reported previously and suggests an association between the length of the first meiotic M phase (MI) and the net synthesis of cyclin B, that seems to regulate the time required for the cdc2 kinase to reach its maximum activity. Moreover, no marked degradation of cyclin B was observed before the MI to MII transition and that which occurs does so independently of the presence of microtubules, which are essential for cyclin degradation during metaphase II arrest and exit of oocytes into interphase of the first mitotic cell cycle. Cyclin B is degraded rapidly during the transitions MI to MII, MII to the first mitotic interphase and MII to an abortive third metaphase state (MIII). However, whilst its degradation was incomplete during the MI to MII transition, virtually no cyclin B protein was detected following both the MII to interphase and MII to MIII transitions. Thus, the decision of oocytes to exit into MIII, or interphase is not controlled at the level of cyclin B degradation. Lastly, in aging, non-activated oocytes, the net synthesis of cyclin B declines. Whereas, in activated eggs cultured in parallel although the rate of net synthesis declines initially, it is effectively ‘rescued’ being two-fold greater than in non-activated oocytes of an equivalent age. This gradual fall in the net synthesis of cyclin B observed in aging oocytes may contribute to the increasing ease with which they become activated, compared to recently ovulated oocytes.     

Mutants of Salmonella typhimurium That Are Insensitive to Catabolite Repression of Proline Degradation

In Salmonella typhimurium the two enzymes of proline catabolism, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid dehydrogenase, are subject to catabolite repression when the cells are grown in the presence of glucose. Mutants partially relieved of catabolite repression (PutR) for the proline catabolic enzymes have been isolated by selection on agar plates containing glucose and proline. The specificity of the catabolite repression-insensitive character for the enzymes of proline utilization has been confirmed by an analysis of other unrelated catabolic enzymes. Histidase and amylomaltase of the mutant strains are equally as sensitive to glucose repression as are the enzymes from the wild type. All four PutR mutants exhibit higher induced and higher basal levels of proline oxidase as compared with the corresponding wild-type levels. The mutations of three strains tested are cotransducible with constitutive, pleiotrophic-negative and structural gene mutations of the put region. Three-factor crosses indicate that two putR mutations are located at one end of the cluster of put mutations.     

Genetic control of autoimmune myocarditis mediated by myosin-specific antibodies

 Autoimmune disease involves both the development of autoreactivity and the expression of organ damage, and susceptibility is genetically complex. We recently reported that in autoimmune myocarditis susceptibility to antibody-mediated cardiac injury is strain specific. DBA/2 mice develop myocarditis following administration of myosin-specific antibody, while BALB/c mice do not. This susceptibility appears to be controlled by expression of myosin in the myocardial extracellular matrix. CByD2F1 mice are both resistant to induction of myocarditis and do not demonstrate extracellular myosin, indicating a recessive genetic component to these traits. A backcross analysis of susceptibility using DBA/2×CByD2F1 mice revealed a locus on chromosome 12 that is strongly linked with myocarditis. In male mice there was a second region on chromosome 1 that also contributes to disease susceptibility. However, genetic susceptibility in both female and male mice was genetically complex. This study demonstrates that the genetic basis of tissue injury can be analyzed separately from the genetic basis of autoreactivity. Future studies will determine whether the genetic factors identified in this study are also involved in susceptibility to rheumatic fever. Received: 18 May 1998 / Revised: 3 July 1998     

Changes in the activity of type 2A protein phosphatases during meiotic maturation and the first mitotic cell cycle in mouse oocytes.

We have established an assay to measure protein phosphatase activity in mouse oocytes using [32P]-radiolabeled phosphorylase a as the substrate. Removal of the radiolabel from the substrate in vitro was linear with time and could be inhibited totally by the addition of okadaic acid (inhibitor of type 1 and type 2 protein phosphatases), or partially by protein inhibitor 2 (inhibitor of type 1 protein phosphatases). We performed a detailed study of the activity of type 2A protein phosphatases in mouse oocytes undergoing meiotic maturation and after parthenogenetic activation of mature oocytes arrested in metaphase II. Significant changes in the activity of type 2A protein phosphatases were observed during the first meiotic and the first mitotic cell cycles. These alterations in type 2A protein phosphatase activity occurred in the absence of changes in the quantity of the catalytic sub-unit and can be correlated with changes in the activity of protein kinases and rearrangement of the cellular cytoskeleton. Our observations support a role for type 2A protein phosphatases in cell cycle regulation and demonstrate that, like the protein kinases, the type 2A phosphatases also undergo changes in their activity during early mammalian development.     

Does zoo herpetology have a future?

Fifty-two North American zoo reptile and amphibian departments were surveyed to determine their contributions to recognized American Zoo and Aquarium Association (AZA)-sponsored programs and formalized research projects over the past 10 years. Surveys also requested information concerning the allocation of resources for conservation and research programs, staff educational background, and entry level salaries. Twenty-two institutions responded to the survey, collectively indicating a total of 164 technical papers, 16 field studies, and 101 non-technical articles completed between 1987 and 1997. Of the 164 technical papers published, 130 (79%) were contributed by three institutions. Of the 16 field studies, seven were outside the United States, whereas nine focused on native species and ecosystems. Six of the reported field studies involved only financial or logistical support. Of the 101 non-technical articles, 42 (42%) were contributed by a single institution. Twenty-one formalized in-house research projects were reported. However, only four appeared to have a clearly defined objective. Survey respondents also reported nine species of reptiles and one amphibian taxon are managed by Species Survival Plans (SSPs). There are currently 12 Taxon Advisory Group (TAG) coordinators, with four of the current coordinators having served on multiple TAG committees. There are 41 AZA-approved studbooks for reptiles and two for amphibians, with 29 having actually been published to date. The average starting salary reported in our survey for entry level keeper positions was $19,500 (range, $13,500–30,000). The average level of education reported was high school graduate. There was no correlation between productivity and higher wages, or level of education. Only one institution received funding specifically for research. We conclude that zoo herpetology departments are not realizing their potential for formalized research and conservation projects and propose recommendations for future involvement. Zoo Biol 17:453–462, 1998. © 1998 Wiley-Liss, Inc.     

DYRK1A stabilizes HPV16E7 oncoprotein through phosphorylation of the threonine 5 and threonine 7 residues

HPV16, a high-risk tumorigenic virus, has been identified as one of the causative agents for the development of cervical cancer. Subsequent to viral infection, the constitutive expression of the viral oncoproteins E6 and E7 plays a number of critical roles in maintaining the transformed phenotype. Here we demonstrate that a cellular kinase, dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), interacts with and phosphorylates HPV16E7 in vitro and in vivo. Using substitution mutations, we identified that DYRK1A specifically phosphorylates HPV16E7 at Thr5 and Thr7, which are located within the N-terminal CRI domain. This interaction greatly increases the steady-state level of HPV-16E7 by interfering with the protein's 26S proteosome-dependent degradation. The half-life of E7 was extended significantly by replacing Thr5 and Thr7 with a phosphorylation mimetic residue, aspartic acid. In addition, DYRK1A-induced phosphorylation protected E7 from degradation and influenced E7's function when modulating pRb degradation. We propose a new mechanism whereby DYRK1A phosphorylates Thr5 and Thr7 within HPV16E7. This phosphorylation then interferes with the degradation of HPV16E7, extending the protein half-life of HPV16E7 and increasing the colony-formation efficacy of HPV16E7. Our findings suggest that DYRK1A increases the transforming potential of HPV16-infected cells because of the greater stability of HPV16E7.