Searched for: school:SOM
Department/Unit:Cell Biology
Cardiolipin biosynthesis and mitochondrial respiratory chain function are interdependent
Gohil, Vishal M; Hayes, Paulette; Matsuyama, Shigemi; Schagger, Hermann; Schlame, Michael; Greenberg, Miriam L
Cardiolipin (CL) is an acidic phospholipid present almost exclusively in membranes harboring respiratory chain complexes. We have previously shown that, in Saccharomyces cerevisiae, CL provides stability to respiratory chain supercomplexes and CL synthase enzyme activity is reduced in several respiratory complex assembly mutants. In the current study, we investigated the interdependence of the mitochondrial respiratory chain and CL biosynthesis. Pulse-labeling experiments showed that in vivo CL biosynthesis was reduced in respiratory complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) and oxidative phosphorylation complex V (ATP synthase) assembly mutants. CL synthesis was decreased in the presence of CCCP, an inhibitor of oxidative phosphorylation that reduces the pH gradient but not by valinomycin or oligomycin, both of which reduce the membrane potential and inhibit ATP synthase, respectively. The inhibitors had no effect on phosphatidylglycerol biosynthesis or CRD1 gene expression. These results are consistent with the hypothesis that in vivo CL biosynthesis is regulated at the level of CL synthase activity by the DeltapH component of the proton-motive force generated by the functional electron transport chain. This is the first report of regulation of phospholipid biosynthesis by alteration of subcellular compartment pH
PMID: 15292198
ISSN: 0021-9258
CID: 49192
Alpha-conotoxin residues that interact at close range with gamma-tyrosine-111 and mutant delta-tyrosine-113 on the Torpedo nicotinic acetylcholine receptor
Velez-Carrasco, Wanda; Valdes, Sonia; Agresar, Lyann; Lettich, Alyssa; Guerra, Astrid Y; Hann, Richard M
The alpha-conotoxins MI and GI display stronger affinities for the alphagamma agonist site on the Torpedo californica electrocyte nicotinic acetylcholine receptor (ACHR) than for the alphadelta agonist site, while alpha-conotoxin SI binds with the same affinity to both sites. Prior studies reported that the arginine at position 9 on GI and the tyrosine at position 111 on the receptor gamma subunit were responsible for the stronger alphagamma affinities of GI and MI, respectively. This study was undertaken to determine if the alpha-conotoxin midchain cationic residues interact with Torpedo gammaY111. The findings show that lysine 10 on MI is responsible for the alphagamma selectivity of MI and confirm the previously reported importance of R9 on GI and on the SI analogue, SIP9R. The results also show that gammaY111 contributes substantially to the selective alphagamma high affinity of all three peptides. Double-mutant cycle analyses reveal that, in the alphagamma site, K10 on MI and R9 on SIP9R interact with the aromatic ring of gammaY111 to stabilize the high-affinity complex, while in contrast, R9 on GI does not. The substitution of Y for R at position 113 on the delta subunit converts the alphadelta site into a high-affinity site for MI, GI, and SIP9R through the interacting of deltaY113 with K10 on MI and with R9 on both GI and SIP9R. The overall data show that the residues in the two sites with which MI interacts, other than at gamma111/delta113, are either the same or similar enough to exert equivalent effects on MI, indicating that MI binds in the same orientation at the alphagamma and alphadelta sites. Similar findings show that SIP9R probably also binds in the same orientation at the wild-type alphagamma and alphadelta sites. The finding that R9 on GI interacts closely with deltaR113Y but not with gammaY111 means that GI binds in different orientations at the alphagamma and alphadelta sites. This report also discusses the molecular basis of the difference in the MI high-affinity sites on Torpedo and embryonic mouse muscle ACHRs.
PMID: 15449960
ISSN: 0006-2960
CID: 173843
Fibrillin microfibrils: multipurpose extracellular networks in organismal physiology
Ramirez, Francesco; Sakai, Lynn Y; Dietz, Harry C; Rifkin, Daniel B
Organismal physiology depends significantly on the proper assembly of extracellular matrix (ECM) macroaggregates that impart structural integrity to the connective tissue. Recent genetic studies in mice have unraveled unsuspected new functions of architectural matrix components in regulating signaling events that modulate patterning, morphogenesis, and growth of several organ systems. As a result, a new paradigm has emerged whereby tissue-specific organization of the ECM dictates not only the physical properties of the connective tissue, but also the ability of the matrix to direct a broad spectrum of cellular activities through the regulation of growth factor signaling. These observations pave the way to novel therapeutic approaches aimed at counteracting the deleterious consequences of perturbations of connective tissue homeostasis.
PMID: 15466717
ISSN: 1094-8341
CID: 710792
Actin-binding proteins in a postsynaptic preparation: Lasp-1 is a component of central nervous system synapses and dendritic spines
Phillips, Greg R; Anderson, Tonya R; Florens, Laurence; Gudas, Christopher; Magda, Gabriela; Yates, John R 3rd; Colman, David R
CNS synapses are complex sites of cell-cell communication. Identification and characterization of the protein components of synapses will lead to a better understanding of the mechanisms of neurotransmission and plasticity. We applied multidimensional protein identification technology (MudPIT) to purified, guanidine-solubilized postsynaptic fractions to identify novel synaptically localized molecules. We identified several actin-associated proteins known to regulate actin polymerization and control cell motility in nonneural cells that have not previously been associated with CNS synaptic function. One of these is lasp-1, an actin-associated LIM and SH3 domain-containing protein. We show that lasp-1 is strongly expressed by CNS neurons and is concentrated at synaptic sites. Overall, the preponderance of actin-associated proteins in postsynaptic density fractions, and specifically those involved in actin reorganization, suggests that there are many modes by which the state of synaptic F-actin polymerization and, hence, synaptic physiology are affected.
PMID: 15372503
ISSN: 0360-4012
CID: 605872
Impact of mandatory resident work hour limitations on medical students' interest in surgery
Miller, George; Bamboat, Zubin M; Allen, Frederick; Biernacki, Peter; Hopkins, Mary Ann; Gouge, Thomas H; Riles, Thomas S
BACKGROUND: The number of US medical students applying for general surgery residency has been declining. Recent studies have shown that the issue of 'controllable lifestyle' has become a critical factor in medical students' decision-making process. We postulate that widespread implementation of resident work hour limitations would bolster medical students' interest in pursuing surgical careers. STUDY DESIGN: Students from New York University School of Medicine were surveyed about their attitudes toward work hour limitations and its effect on their interest in pursuing a surgical residency. One hundred thirty-two students participated. RESULTS: Nearly 95% of respondents believed that work hour limitations were a positive change and, if all other factors were equal, they would choose a training program that used work hour limitations over one that did not. The most common reasons cited in favor of limits were improvements in resident lifestyle (42%) and patient safety (34%). Fifty-three percent of respondents indicated that presence of work hour limitations alone would increase their interest in considering a surgical residency and only 2% of medical students indicated that it would lessen their interest in surgery. Not surprisingly, intellectual interest in a specialty was the most important factor in choosing a residency for 86% of students. Nevertheless, work hour limitations were designated a higher priority than future salary by 55% of medical students. CONCLUSIONS: The presence of work hour limitations has a positive impact on medical students' interest in surgery. Widespread implementation of work hour limitations may bolster the number of applications for surgical residency
PMID: 15454148
ISSN: 1072-7515
CID: 46084
Quantitative image analysis in mammary gland biology
Fernandez-Gonzalez, Rodrigo; Barcellos-Hoff, Mary Helen; Ortiz-de-Solorzano, Carlos
In this paper we present a summary of recent quantitative approaches used for the analysis of macro and microscopic images in mammary gland biology. The advantages and disadvantages of whole mount analysis, reconstruction of serial tissue sections and nucleus/cell segmentation of either conventional and confocal images are discussed, as are applications of quantitative image analysis, such as quantification of protein levels or vasculature measurements in normal tissue and cancer. Integration of quantitative imaging into the further study of the mammary gland holds the promise of better understanding its tissue complexity that evolves during development, differentiation and disease
PMID: 15838604
ISSN: 1083-3021
CID: 83198
The Tyr (albino) locus of the laboratory mouse
Beermann, Friedrich; Orlow, Seth J; Lamoreux, M Lynn
The albino mouse was already known in ancient times and was apparently selectively bred in Egypt, China, and Japan. Thus, it is not surprising that the c or albino locus (now the Tyr locus) was among the first used to demonstrate Mendelian inheritance in mammals at the dawn of the past century. This locus is now known to encode tyrosinase, the rate-limiting enzyme in the production of melanin pigment, and the molecular basis of the albino ( Tyr(c)) mutation is known. Here we describe the congenic series of Tyr-locus alleles, from wild type to null ( albino). We compare eye and skin pigmentation phenotypes and the genetic lesions that cause each. We suggest that this panel of congenic mutants contains rich, untapped resources for the study of many questions of basic cell biological interest
PMID: 15520878
ISSN: 0938-8990
CID: 49631
C/EBP homologous protein (CHOP, Ddit3) is essential for osteoblastic function [Meeting Abstract]
Pereira, RC; Marciniak, SJ; Ron, D; Canalis, E
ISI:000224326800054
ISSN: 0884-0431
CID: 56283
Alzheimer amyloid precursor aspartyl proteinase activity in CHAPSO homogenates of Spodoptera frugiperda cells
Carter, Troy L; Verdile, Giuseppe; Groth, David; Bogush, Alexey; Thomas, Stefani; Shen, Patrick; Fraser, Paul E; Mathews, Paul; Nixon, Ralph A; Ehrlich, Michelle E; Kwok, John B J; St George-Hyslop, Peter; Schofield, Peter; Li, Yueming; Yang, Austin; Martins, Ralph N; Gandy, Sam
Presenilins are polytopic, integral proteins that control intramembranous proteolysis at the 'gamma-' and 'epsilon-' cleavage sites of the Alzheimer amyloid-beta precursor protein (APP) to yield amyloid-beta peptide (Abeta) and the APP intracellular domain (AICD). We have overexpressed a constitutively active, pathogenic form of PS1 (known as PS1 Delta exon 9) together with its substrate, APP-C99, in Spodoptera frugiperda (Sf9) cells. Sf9 cells have been reported to lack endogenous gamma-secretase, an unexpected finding since there exists an insect homologue of PS1. In our hands, neither intact insect cells coexpressing PS1 Delta exon 9/APP-C99 nor the aqueous homogenates of these cells displayed obvious products of the gamma- or epsilon-secretase reactions, as reported. Surprisingly, when APP-C99-expressing cells were homogenized in 3[(3-cholamidopropyl) dimethylammonio]-2-hydroxypropanesulfonic acid (CHAPSO), a detergent known to support gamma-secretase activity, subsequent incubation led to the accumulation of an AICD-like peptide (AICD-L). Aspartyl proteinase inhibitors were effective in preventing the appearance of AICD-L, but inhibitors of other classes of proteinases were ineffective. Immunoprecipitation-mass spectrometry of AICD-L revealed its identity as the minor of the two known AICDs
PMID: 15592142
ISSN: 0893-0341
CID: 55765
Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM
Sengupta, Jayati; Nilsson, Jakob; Gursky, Richard; Spahn, Christian M T; Nissen, Poul; Frank, Joachim
RACK1 serves as a scaffold protein for a wide range of kinases and membrane-bound receptors. It is a WD-repeat family protein and is predicted to have a beta-propeller architecture with seven blades like a Gbeta protein. Mass spectrometry studies have identified its association with the small subunit of eukaryotic ribosomes and, most recently, it has been shown to regulate initiation by recruiting protein kinase C to the 40S subunit. Here we present the results of a cryo-EM study of the 80S ribosome that positively locate RACK1 on the head region of the 40S subunit, in the immediate vicinity of the mRNA exit channel. One face of RACK1 exposes the WD-repeats as a platform for interactions with kinases and receptors. Using this platform, RACK1 can recruit other proteins to the ribosome
PMID: 15334071
ISSN: 1545-9985
CID: 66319