Jeffrey M.C. Lau

Plus and Minus Sexual Agglutinins from Chlamydomonas reinhardtii

Gametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins. Both agglutinin proteins are organized into three distinct domains: a head, a shaft in a polyproline II configuration, and an N-terminal domain. The plus and minus heads are related in overall organization but poorly conserved in sequence except for two regions of predicted hydrophobic α-helix. The shafts contain numerous repeats of the PPSPX motif previously identified in Gp1, a cell wall HRGP. We propose that the head domains engage in autolectin associations with the distal termini of their own shafts and suggest ways that adhesion may involve head–head interactions, exolectin interactions between the heads and shafts of opposite type, and antiparallel shaft–shaft interactions mediated by carbohydrates displayed in polyproline II configurations.

The 14-3-3τ phosphoserine-binding protein is required for cardiomyocyte survival

ABSTRACT 14-3-3 family members are intracellular dimeric phosphoserine-binding proteins that regulate signal transduction, cell cycle, apoptotic, and metabolic cascades. Previous work with global 14-3-3 protein inhibitors suggested that these proteins play a critical role in antagonizing apoptotic cell death in response to provocative stimuli. To determine the specific role of one family member in apoptosis, mice were generated with targeted disruption of the 14-3-3τ gene. 14-3-3τ−/− mice did not survive embryonic development, but haploinsufficient mice appeared normal at birth and were fertile. Cultured adult cardiomyocytes derived from 14-3-3τ+/− mice were sensitized to apoptosis in response to hydrogen peroxide or UV irradiation. 14-3-3τ+/− mice were intolerant of experimental myocardial infarction and developed pathological ventricular remodeling with increased cardiomyocyte apoptosis. ASK1, c-jun NH2-terminal kinase, and p38 mitogen-activated protein kinase (MAPK) activation was increased, but extracellular signal-regulated kinase MAPK activation was reduced, in 14-3-3τ+/− cardiac tissue. Inhibition of p38 MAPK increased survival in 14-3-3τ+/− mice subjected to myocardial infarction. These results demonstrate that 14-3-3τ plays a critical antiapoptotic function in cardiomyocytes and that therapeutic agents that increase 14-3-3τ activity may be beneficial to patients with myocardial infarction.

Differential role of 14-3-3 family members in Xenopus development.

The 14‐3‐3 proteins are intracellular dimeric phosphoserine/threonine binding molecules that participate in signal transduction, checkpoint control, nutrient sensing, and cell survival pathways. Previous work established that 14‐3‐3 proteins are required in early Xenopus laevis development by modulating fibroblast growth factor signaling. Although this general requirement for 14‐3‐3 proteins in Xenopus early embryogenesis is established, there is no information about the specific role of individual 14‐3‐3 genes. Botanical studies previously demonstrated functional specificity among 14‐3‐3 genes during plant development. In this study, an antisense morpholino oligo microinjection approach was used to characterize the requirement for six specific 14‐3‐3 family members in Xenopus embryogenesis. Microinjection experiments followed by Western blot analysis showed that morpholinos reduced specific 14‐3‐3 protein levels. Embryos lacking specific 14‐3‐3 isoforms displayed unique phenotypic defects. In particular, reduction of 14‐3‐3 tau (τ) protein, and to a lesser extent, 14‐3‐3 epsilon (ϵ), resulted in embryos with prominent gastrulation and axial patterning defects and reduced mesodermal marker gene expression. In contrast, reduction of 14‐3‐3 zeta (ζ) protein caused no obvious phenotypic abnormalities. Reduction of 14‐3‐3 gamma (γ) protein resulted in eye defects without gastrulation abnormalities. Therefore, individual 14‐3‐3 genes have separable functions in vertebrate embryonic development. Developmental Dynamics 235:1761–1776, 2006.

Differential functions of 14-3-3 isoforms in vertebrate development.

Publisher Summary The 14-3-3 proteins are intracellular dimeric phosphoserine/threonine-binding molecules that are found in all eukaryotic organisms. It participates in developmental, signal transduction, checkpoint control, nutrient sensing, and cell survival pathways. In fungi, plants, and animals, there are multiple 14-3-3 proteins encoded by separate genes. This chapter discusses the roles of 14-3-3 family members in animal development. Individual vertebrate 14-3-3 genes exhibit differential expression patterns and may have unique binding partners. Initial experiments suggest that each 14-3-3 protein may specifically regulate different aspects of vertebrate development. The chapter also discusses the structure and regulation of 14-3-3 proteins. The molecular interference model explains the action of 14-3-3 proteins on a variety of binding partners. The role of 14-3-3 proteins in the development of caenorrhabditis elegans is well studied by the analysis of mutants and use of RNAi. There are two 14-3- 3 genes in worms, ftt-1 (par-5) and ftt2. The ftt-1 gene is highly expressed in early embryos as maternal transcript in a symmetrical pattern, but expression decreases markedly in later development. In contrast, ftt-2 is undetectable in 1-cell embryos and is first detected in 2-cell or 4-cell embryos, suggesting that it is an early zygotic transcript. The ftt-2 gene is expressed at high levels in the posterior region of post-proliferative embryos. The unique functional roles of 14-3-3 isoforms in vertebrate development may be due to differential patterns of gene expression, subcellular localization, regulation of activity, or ability to bind to target proteins. In Xenopus development, the asymmetric localization of 14-3-3 ɛ protein clearly seems to underlie its function in the development of left–right asymmetry.

Feasibility of MRI attenuation correction in cardiac FDG-PET

Background Simultaneous acquisition PET-MRI is a new technology that has the potential to significantly impact diagnostic patient care. Cardiac imaging using PET-MRI offers high signal resolution MRI images superimposed on PET metabolic functional assessment. Specifically, 18Ffluorodeoxyglucose (FDG) PET-MR has the potential to provide both anatomic scar tissue evaluation and information regarding myocardial glucose metabolism. While early brain and soft tissue data have demonstrated that PET specific uptake values (SUVs) obtained using MRI for attenuation correction (AC) are comparable to SUVs obtained using CT AC, SUV measurements of myocardial tissue have not been compared. The objective of this pilot study is to determine the reproducibility of SUVs obtained by PET imaging using an AC µ-map comprised of a dual echo VIBE Dixon MRI sequence instead of CT. Methods

Demonstration of intermittent ischemia and stunning in hibernating myocardium

Intermittent ischemia and stunning appear to characterize hibernating myocardium. In this report, we demonstrate the variable flow abnormalities that may underlie these phenomena. A 72-year-old woman underwent a rest/vasodilator stress ECG-gated Tc-SPECT myocardial perfusion imaging (MPI). Image interpretation was consistent with a large anterior/anteroapical infarction with borderzone ischemia (Figure 1). Three days later, repeat rest/vasodilator MPI using N-ammonia and late gadolinium enhancement imaging (LGE) was performed simultaneously on a PET/MR (Biograph mMR, Siemens) as part of a research protocol to compare the accuracy of ischemia detection between SPECT and PET/MR. PET/MR MPI revealed extensive and severe anterior/anteroapical ischemia without infarction with resting and post-stress anteroapical hypokinesis (Figure 2A, B; Supplementary Movies 13). LGE PET/MR images showed no evidence of infarction in the area of ischemia detected by PET/MR (Figure 2C-E). The constellation of these findings allowed us to speculate that there was myocardial hibernation. Of note, absolute MBF calculated from the PET data demonstrated near similar resting flow values between the hibernating and normal myocardium with a paradoxical reduction in flow in the hibernating territory at stress, indicative coronary steal (Figure 3). Subsequent coronary angiography confirmed high grade LAD disease (Figure 4). This case provides evidence of the various types of flow abnormalities that characterize myocardial hibernation. They include resting hypoperfusion, impaired vasodilator capacity, and coronary steal. These flow abnormalities, either individually or collectively, can result in myocardial ischemia. However, their intermittent nature will lead to myocardial stunning (normal resting perfusion with abnormal resting wall motion). The finding of variable resting flow values based on the differences in the relative SPECT and PET/MR MPI studies is particularly noteworthy as it may impact the accuracy of SPECT and PET viability studies that are based on relative flow assessment.

Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 tau exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 gamma exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 zeta appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

Cardiac Applications of PET/MR Imaging

Simultaneous acquisition PET/MR imaging combines the anatomic capabilities of cardiac MR imaging with quantitative capabilities of both PET and MR imaging. Cardiac PET/MR imaging has the potential not only to assess cardiac tumors but also to provide thorough assessment of myocardial ischemia, infarction, and function and specific characterization of cardiomyopathies, such as cardiac sarcoid. In this article, the authors start with a discussion of the technical challenges specific to cardiovascular PET/MR imaging followed by a discussion of the use of PET/MR imaging in various cardiovascular conditions.