A novel metabolic form of the 32 kDa-D1 protein in the grana-localized reaction center of photosystem II

Two forms of the 32 kDa-D1 reaction center protein of photosystem II (PSII), having slightly different mobilities on denaturing polyacrylamide gels, have been resolved in Spirodela oligorrhiza, Glycine max L., Gossypium hirsutum L., Triticum aestivum L., and Zea mays L. The protein band with faster mobility is identified as the 32 kDa-D1 protein, and the less mobile band as a novel form, designated 32*. The two forms are structurally similar based on immunological and partial proteolytic tests. 32* is associated exclusively with the grana and is present in the PSII reaction center. Temporally, 32* appears several hours after the translocation of newly synthesized and processed 32 kDa-D1 protein from the stroma lamellae to the grana. Formation of the 32* is strictly light-dependent under physiological light intensities and correlates with a reciprocal loss of the 32-kDa form. Light induced formation of 32* is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is not coupled to linear electron transport.     

Rapid chromosome territory relocation by nuclear motor activity in response to serum removal in primary human fibroblasts

Background  

Radial chromosome positioning in interphase nuclei is nonrandom and can alter according to developmental, differentiation, proliferation, or disease status. However, it is not yet clear when and how chromosome repositioning is elicited.     

Analytical errors in measuring radioactivity in cell proteins and their effect on estimates of protein turnover in L cells.

Previous studies from this laboratory on protein turnover in 3H-labelled L-cell cultures have shown recovery of total 3H at the end of a 3-day experiment to be always significantly in excess of the 3H recovered at the beginning of the experiment. In this study we have critically reviewed a number of possible sources for this error in measuring radioactivity in cell proteins. 3H-labelled proteins, when dissolved in 0.3 M-NaOH and counted for radioactivity in a liquid-scintillation spectrometer, showed losses of 30-40% of the radioactivity; neither external or internal standardization compensated for this loss. Hydrolysis of these proteins with either Pronase or concentrated HCl significantly increased the measured radioactivity. In addition, approx. 5-10% of the cell protein is left on the plastic culture dish when cells are recovered in phosphate-buffered saline. To aggravate this latter loss further, this surface-adherent protein, after pulse labelling, contains proteins of high radioactivity that turn over rapidly and make a major contribution to the accumulating radioactivity in the medium. These combined errors can account for up to 60% of the total radioactivity in the cell culture. Similar analytical errors have been found in studies of other cell cultures. The effect of these analytical errors on estimates of protein turnover in cell cultures is discussed.     

Crystal Structure of the cGMP-dependent Protein Kinase II Leucine Zipper and Rab11b Protein Complex Reveals Molecular Details of G-kinase-specific Interactions

cGMP-dependent protein kinase (PKG)-interacting proteins (GKIPs) mediate cellular targeting of PKG isoforms by interacting with their leucine zipper (LZ) domains. These interactions prevent aberrant signaling cross-talk between different PKG isotypes. To gain detailed insight into isotype-specific GKIP recognition by PKG, we analyzed the type II PKG leucine zipper domain and found that residues 40–83 dimerized and specifically interacted with Rab11b. Next, we determined a crystal structure of the PKG II LZ-Rab11b complex. The PKG II LZ domain presents a mostly nonpolar surface onto which Rab11b docks, through van der Waals interactions. Contact surfaces in Rab11b are found in switch I and II, interswitch, and the β1/N-terminal regions. This binding surface dramatically differs from that seen in the Rab11 family of interacting protein complex structures. Structural comparison with PKG Iα and Iβ LZs combined with mutagenic analysis reveals that GKIP recognition is mediated through surface charge interactions.     

A Biodegradable,Sustained-Released,Prednisolone Acetate Microfilm Drug Delivery System Effectively Prolongs Corneal Allograft Survival in the Rat Keratoplasty Model

Frequent and long-term use of topical corticosteroids after corneal transplantation is necessary to prevent graft rejection. However, it relies heavily on patient compliance, and sustained therapeutic drug levels are often not achieved with administration of topical eye drops. A biodegradable drug delivery system with a controlled and sustained drug release may circumvent these limitations. In this study, we investigated the efficacy of a prednisolone acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC) microfilm drug delivery system on promoting the survival of allogeneic grafts after penetrating keratoplasty (PK) using a rat model. The drug release profiles of the microfilms were characterized (group 1). Subsequently, forty-eight PK were performed in four experimental groups: syngeneic control grafts (group 2), allogeneic control grafts (group 3), allogeneic grafts with subconjunctivally-implanted PA microfilm (group 4), and allogeneic grafts with PA eye drops (group 5; n = 12 in each). PA-loaded microfilm achieved a sustained and steady release at a rate of 0.006–0.009 mg/day, with a consistent aqueous drug concentration of 207–209 ng/ml. The mean survival days was >28 days in group 2, 9.9±0.8 days in group 3, 26.8±2.7 days in group 4, and 26.4±3.4 days in group 5 (P = 0.023 and P = 0.027 compared with group 3). Statistically significant decrease in CD4+, CD163+, CD 25+, and CD54+ cell infiltration was observed in group 4 and group 5 compared with group 3 (P<0.001). There was no significant difference in the mean survival and immunohistochemical analysis between group 4 and group 5. These results showed that sustained PA-loaded microfilm effectively prolongs corneal allograft survival. It is as effective as conventional PA eye drops, providing a promising clinically applicable alternative for patients undergoing corneal transplantation.