Reversible phosphorylation and turnover of the D1 protein under various redox states of Photosystem II induced by low temperature photoinhibition

Reversible phosphorylation and turnover of the D1 protein in vivo were studied under low-temperature photoinhibition of pumpkin leaves and under subsequent recovery at low light at 4 °C or 23 °C. The inactivation of PS II and photodamage to D1 were not enhanced during low-temperature photoinhibition when compared to that at room temperature. The PS II repair cycle, however, was completely blocked at 4 °C at the level of D1 degradation. Both the recovery of the photochemical activity of PS II and the degradation of the damaged D1 protein at low light at 23 °C were delayed about 1 hour after low-temperature photoinhibition, suggesting that in addition to the decrease in catalytic turnover of the enzyme, the protease was specifically inactivated in vivo at low temperature. The effect of low temperature on the other regulatory enzymes of PS II repair, protein kinase and phosphatase [Rintamäki et al. (1996) J Biol Chem 271: 14870-14875] was variable. The D1 protein kinase was operational at low temperature while dephosphorylation of the D1 protein seemed to be completely inhibited during low temperature treatment. Under subsequent recovery conditions at low light and 23 °C, the high phosphorylation level of D1 was sustained in leaf discs photoinhibited at low temperature, despite the recovery of the phosphatase activity. This high phosphorylation level of D1 was due to the persistently active kinase. The D1 kinase, previously shown to get activated by reduction of plastoquinone, was, however, found to be maximally active already at relatively low redox state of the plastoquinone pool. We suggest that phosphorylation of PS II centers increases the stability of PS II complexes and concomitantly improves their survival under stress conditions.     

ADP Compartmentation Analysis Reveals Coupling between Pyruvate Kinase and ATPases in Heart Muscle

Cardiomyocytes have intracellular diffusion restrictions, which spatially compartmentalize ADP and ATP. However, the models that predict diffusion restrictions have used data sets generated in rat heart permeabilized fibers, where diffusion distances may be heterogeneous. This is avoided by using isolated, permeabilized cardiomyocytes. The aim of this work was to analyze the intracellular diffusion of ATP and ADP in rat permeabilized cardiomyocytes. To do this, we measured respiration rate, ATPase rate, and ADP concentration in the surrounding solution. The data were analyzed using mathematical models that reflect different levels of cell compartmentalization. In agreement with previous studies, we found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases in the cell. In addition, our experimental data show that considerable activity of endogenous pyruvate kinase (PK) remains in the cardiomyocytes after permeabilization. A fraction of ATPases were inactive without ATP feedback by this endogenous PK. When analyzing the data, we were able to reproduce the measurements only with the mathematical models that include a tight coupling between the fraction of endogenous PK and ATPases. To our knowledge, this is the first time such a strong coupling of PK to ATPases has been demonstrated in permeabilized cardiomyocytes.     

Development of FuGO: an ontology for functional genomics investigations

The development of the Functional Genomics Investigation Ontology (FuGO) is a collaborative, international effort that will provide a resource for annotating functional genomics investigations, including the study design, protocols and instrumentation used, the data generated and the types of analysis performed on the data. FuGO will contain both terms that are universal to all functional genomics investigations and those that are domain specific. In this way, the ontology will serve as the "semantic glue" to provide a common understanding of data from across these disparate data sources. In addition, FuGO will reference out to existing mature ontologies to avoid the need to duplicate these resources, and will do so in such a way as to enable their ease of use in annotation. This project is in the early stages of development; the paper will describe efforts to initiate the project, the scope and organization of the project, the work accomplished to date, and the challenges encountered, as well as future plans.     

Loss of SUFU Function in Familial Multiple Meningioma

Meningiomas are the most common primary tumors of the CNS and account for up to 30% of all CNS tumors. An increased risk of meningiomas has been associated with certain tumor-susceptibility syndromes, especially neurofibromatosis type II, but no gene defects predisposing to isolated familial meningiomas have thus far been identified. Here, we report on a family of five meningioma-affected siblings, four of whom have multiple tumors. No NF2 mutations were identified in the germline or tumors. We combined genome-wide linkage analysis and exome sequencing, and we identified in suppressor of fused homolog (Drosophila), SUFU, a c.367C>T (p.Arg123Cys) mutation segregating with the meningiomas in the family. The variation was not present in healthy controls, and all seven meningiomas analyzed displayed loss of the wild-type allele according to the classic two-hit model for tumor-suppressor genes. In silico modeling predicted the variant to affect the tertiary structure of the protein, and functional analyses showed that the activity of the altered SUFU was significantly reduced and therefore led to dysregulated hedgehog (Hh) signaling. SUFU is a known tumor-suppressor gene previously associated with childhood medulloblastoma predisposition. Our genetic and functional analyses indicate that germline mutations in SUFU also predispose to meningiomas, particularly to multiple meningiomas. It is possible that other genic mutations resulting in aberrant activation of the Hh pathway might underlie meningioma predisposition in families with an unknown etiology.     

Book Reviews

Boehm, G. and Leuschner, R. M. (eds.). 1987. Advances in Aerobiology. Proceedings of the 3rd International Conference on Aerobiology, August 6–9, 1986, Basel, Switzerland. Experientia Supplementum, Vol. 51, Birkhäuser Verlag, Basel, Boston, 437 pp. ISBN 1803–1. SFr. 98.00.

Nair, P. K. K., Joshi, A. P. and Gangal, S. V. (eds.) 1986. Airborne Pollen, Spores and Other Plant Materials of India. — A Survey. Published jointly by CSIR Centre for Biochemicals and National Botanic Research Institute Lucknow, 224 pp.     

rDNA-directed integration by an HIV-1 integrase—I-PpoI fusion protein

Integrating viral vectors are efficient gene transfer tools, but their integration patterns have been associated with genotoxicity and oncogenicity. The recent development of highly specific designer nucleases has enabled target DNA modification and site-specific gene insertion at desired genomic loci. However, a lack of consensus exists regarding a perfect genomic safe harbour (GSH) that would allow transgenes to be stably and reliably expressed without adversely affecting endogenous gene structure and function. Ribosomal DNA (rDNA) has many advantages as a GSH, but efficient means to target integration to this locus are currently lacking. We tested whether lentivirus vector integration can be directed to rDNA by using fusion proteins consisting of the Human Immunodeficiency Virus 1 (HIV-1) integrase (IN) and the homing endonuclease I-PpoI, which has natural cleavage sites in the rDNA. A point mutation (N119A) was introduced into I-PpoI to abolish unwanted DNA cleavage by the endonuclease. The vector-incorporated IN-I-PpoI_N119A fusion protein targeted integration into rDNA significantly more than unmodified lentivirus vectors, with an efficiency of 2.7%. Our findings show that IN-fusion proteins can be used to modify the integration pattern of lentivirus vectors, and to package site-specific DNA-recognizing proteins into vectors to obtain safer transgene integration.     

In vitro maturation supplements affect developmental competence of bovine cumulus-oocyte complexes and embryo quality after vitrification

Oocyte quality affects subsequent embryo development and quality. We examined the impact of bovine oocyte in vitro maturation (IVM) conditions on subsequent embryo yield, quality and cryosurvival. Cumulus–oocyte complexes (COCs) were sampled for cytological and gene expression analysis after IVM in TCM199 supplemented with 10% fetal bovine serum (FBS), 4 mg/ml of fatty-acid-free bovine serum albumin (FAFBSA), 4 mg/ml of polyvinylpyrrolidone (PVP), FAFBSA with epidermal growth factor (EGF, 100 ng/ml) and insulin-like growth factor 1 (IGF-I, 100 ng/ml) (FAFBSAGF), PVP with EGF and IGF-I (PVPGF) or PVP with single strength BME and MEM amino acids (PVPAA). The remaining COCs were fertilized. On day 7 (IVF = day 0) quality 1 blastocysts were vitrified or analyzed for glucose transporter 1 (Glut-1) expression levels. The remaining blastocysts (days 7–9) were evaluated for morphology and total cell counts. After warming, survival and hatching rates were evaluated followed by total cell counts and Glut-1 expression levels. Only PVPGF IVM resulted in embryo production rates comparable to those recorded with FBS IVM. Growth factors with FAFBSA and amino acids with PVP reduced embryo production rates whereas the effect of the growth factors with PVP was negligible. Insulin-like growth factor 2 binding protein 3 (IGF2BP3) and beta cell translocation gene 4 (BTG4) were revealed as potential candidates for oocyte developmental competence, and secreted protein, acidic and rich in cysteine (SPARC) for cumulus cell expansion. There were no differences among treatments in hatching rates of vitrified embryos after warming. However, total cell numbers and Glut-1 expression levels at 72 h were affected.