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1.  Assessment of Choroidal Thickness and Volume During the Water Drinking Test By Swept-Source Optical Coherence Tomography 
Ophthalmology  2013;120(12):10.1016/j.ophtha.2013.07.040.
To evaluate changes in peripapillary and macular choroidal thickness and volume after the water-drinking test (WDT) using swept-source optical coherence tomography (SS-OCT).
Prospective, cross-sectional observational study.
Fifty-six eyes of 28 healthy volunteers.
Participants underwent a 3-dimensional optic disc and macula scanning protocol with a prototype SS-OCT (Topcon Inc., Tokyo, Japan) at baseline and 15, 30, 45, and 120 minutes after the start of the WDT. The WDT consisted of drinking 1000mL of water within five minutes. Objective measurements of the choroid were obtained with automated segmentation of the choroidal boundaries.
Main Outcome Measures
Choroidal thickness and volume.
Mean (standard deviation) age of participants was 35.6 ± 9.1 years. Intraocular pressure (IOP) increased from 14.9 ± 2.7 mmHg at baseline to a peak of 16.8 ± 3.0 mmHg at 15 minutes after the WDT (p<0.001). Mean baseline choroidal thickness and volume were 181.3 ± 50.8 μm and 6.19 ± 1.80 mm3 at the optic disc and 217.4 ± 43.6 μm and 7.83 ± 1.55 mm3 at the macula. Following the WDT, peripapillary and macular choroidal thickness increased by a maximum of 5.7% (P < 0.001) and 4.3% (P < 0.001) respectively. Choroidal volumes increased by 6.4% (P < 0.001) and 3.9% (P < 0.001), respectively. There was no association between change in IOP and peripapillary (P = 0.27) or macular (P = 0.09) choroidal thickness.
Using automated segmentation of SS-OCT measurements, significant increases in choroidal thickness and volume are observed after the WDT in healthy subjects.
PMCID: PMC3833954  PMID: 24021895
2.  Glutathione S-Transferases Interact with AMP-Activated Protein Kinase: Evidence for S-Glutathionylation and Activation In Vitro 
PLoS ONE  2013;8(5):e62497.
AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.
PMCID: PMC3669356  PMID: 23741294
3.  Declines in swimming performance with age: a longitudinal study of Masters swimming champions 
Because of its many participants and thorough records, competitive Masters swimming offers a rich data source for determining the rate of physical decline associated with aging in physically fit individuals. The decline in performance among national champion swimmers, both men and women and in short and long swims, is linear, at about 0.6% per year up to age 70–75, after which it accelerates in quadratic fashion. These conclusions are based primarily on cross-sectional studies, and little is known about individual performance declines with aging. Herein we present performance profiles of 19 male and 26 female national and international champion Masters swimmers, ages 25 to 96 years, participating in competitions for an average of 23 years.
Methods and results
Swimmers’ longitudinal data were compared with the fastest times of world record holders across ages 35–100 years by two regression methods. Neither method proved to accurately model this data set: compared with the rates of decline estimated from the world record data, which represent the best recorded times at given ages, there was bias toward shallower rates of performance decline in the longitudinal data, likely owing to a practice effect in some swimmers as they began their Masters programs. In swimmers’ later years, once maximum performance had been achieved, individual profiles followed the decline represented in the world records, and a few swimmers became the world record holders. In some instances, the individual profiles indicated performance better than the world record data; these swimmers achieved their times after the world record data were collected in 2005–2006.
Declining physiological functional capacity occurs with advancing age, and this is reflected in the performance decrements of aging Masters swimmers. Individual swimmers show different performance trajectories with aging, declines being mitigated by practice, which improves both physiological capacity and swimming technique, particularly in the early years of participation. The longitudinal data of this study indicate that individuals can participate in high-intensity swimming over several decades, competitively improving over those decades until, in some instances, they become world record holders for their age groups.
PMCID: PMC3871048  PMID: 24379710
physiological functional capacity; geriatric health
4.  The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases 
Nature cell biology  2010;12(12):1177-1185.
Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing N-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 co-target an internal degron of the Mgt1 DNA repair protein. Ufd4 is an E3 of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we report that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1-Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1-Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately over two decades.
PMCID: PMC3003441  PMID: 21076411
proteolysis; polyubiquitin; ubiquitylation; yeast; Saccharomyces cerevisiae
5.  Yeast Two-Hybrid, a Powerful Tool for Systems Biology 
A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided.
PMCID: PMC2705515  PMID: 19582228
interactomics; mass spectrometry; protein-protein interaction; systems bioenergetics; yeast two-hybrid
6.  Indications for a Novel Muscular Dystrophy Pathway 
The Journal of Cell Biology  2000;151(2):235-248.
γ-Filamin, also called ABP-L, is a filamin isoform that is specifically expressed in striated muscles, where it is predominantly localized in myofibrillar Z-discs. A minor fraction of the protein shows subsarcolemmal localization. Although γ-filamin has the same overall structure as the two other known isoforms, it is the only isoform that carries a unique insertion in its immunoglobulin (Ig)-like domain 20. Sequencing of the genomic region encoding this part of the molecule shows that this insert is encoded by an extra exon. Transient transfections of the insert-bearing domain in skeletal muscle cells and cardiomyocytes show that this single domain is sufficient for targeting to developing and mature Z-discs. The yeast two-hybrid method was used to identify possible binding partners for the insert-bearing Ig-like domain 20 of γ-filamin. The two Ig-like domains of the recently described α-actinin–binding Z-disc protein myotilin were found to interact directly with this filamin domain, indicating that the amino-terminal end of γ-filamin may be indirectly anchored to α-actinin in the Z-disc via myotilin. Since defects in the myotilin gene were recently reported to cause a form of autosomal dominant limb-girdle muscular dystrophy, our findings provide a further contribution to the molecular understanding of this disease.
PMCID: PMC2192634  PMID: 11038172
filamin; myotilin; limb-girdle muscular dystrophy; myofibrils; Z-disc proteins
7.  Different Domains of the M-Band Protein Myomesin Are Involved in Myosin Binding and M-Band TargetingV⃞ 
Molecular Biology of the Cell  1999;10(5):1297-1308.
Myomesin is a 185-kDa protein located in the M-band of striated muscle where it interacts with myosin and titin, possibly connecting thick filaments with the third filament system. By using expression of epitope-tagged myomesin fragments in cultured cardiomyocytes and biochemical binding assays, we could demonstrate that the M-band targeting activity and the myosin-binding site are located in different domains of the molecule. An N-terminal immunoglobulin-like domain is sufficient for targeting to the M-band, but solid-phase overlay assays between individual N-terminal domains and the thick filament protein myosin revealed that the unique head domain contains the myosin-binding site. When expressed in cardiomyocytes, the head domains of rat and chicken myomesin showed species-specific differences in their incorporation pattern. The head domain of rat myomesin localized to a central area within the A-band, whereas the head domain of chicken myomesin was diffusely distributed in the cytoplasm. We therefore conclude that the head domain of myomesin binds to myosin but that this affinity is not sufficient for the restriction of the domain to the M-band in vivo. Instead, the neighboring immunoglobulin-like domain is essential for the precise incorporation of myomesin into the M-band, possibly because of interaction with a yet unknown protein of the sarcomere.
PMCID: PMC25262  PMID: 10233145

Results 1-7 (7)