Mode of Action of Nuclease P1 on Nucleic Acids and Its Specificity for Synthetic Phosphodiesters

Nuclease P₁ was found to attack RNA and heat-denatured DNA in endo- and exonucleolytic manners. The evidence was as follows: (1) In the early stage of digestion both mononucleotides and oligonucleotides with various sizes were formed simultaneously with rapid fragmentation of polynucleotides. (2) The relative amount of the monomer was larger than that of any class of oligomers throughout the process of digestion. Nuclease P₁ showed a preference for the linkages between 3′-hydroxyl group of adenosine or deoxyadenosine and the 5′-phosphoryl group of the adjacent nucleotides. p-Nitrophenyl ester of 3′-dTMP was hydrolyzed to thymidine and p-nitrophenyl phosphate, while p-nitrophenyl ester of 5′-dTMP was not attacked. It is concluded from these findings that the basic structure required for the substrate of nuclease P₁ is a nucleoside 3′-phosphate-containing structure and the enzyme cleaves the diester bond between the phosphate and the 3′-hydroxyl group of the sugar.     

Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase

Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity.     

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer ¹⁸F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (¹⁸F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using ¹⁸F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using ¹⁸F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10–40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment ¹⁸F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate ¹⁸F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced ¹⁸F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment ¹⁸F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.     

Shigella Mediated Depletion of Macrophages in a Murine Breast Cancer Model Is Associated with Tumor Regression

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TΔaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TΔaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression.These data support TAMs as useful targets for antitumor therapy and highlight attenuated bacterial pathogens as potential tools.     

Using 31P NMR Spectroscopy at 14.1 Tesla to Investigate PARP-1 Associated Energy Failure and Metabolic Rescue in Cerebrocortical Slices

PARP-1 activation by H(2)O(2) in an acute preparation of superfused, respiring, neonatal cerebrocortical slices was assessed from PAR-polymer formation detected with immunohistochemistry and Western blotting. (31)P NMR spectroscopy at 14.1 Tesla of perchloric acid slice extracts was used to assess energy failure in a 1-h H(2)O(2) exposure as well as in a subsequent 4-h recovery period where the superfusate had no H(2)O(2) and specifically chosen metabolic substrates. Although more data are needed to fully characterize different bioenergetic responses, a high NMR spectral resolution (PCr full-width at half-max approximately.01 ppm) and narrow widths for most metabolites (<.2 ppm) permitted accurate quantifications of spectrally resolved resonances for ADP, ATP, NAD(+)/NADH, and other high energy phosphates. It appears possible to use brain slices to quantitatively study PARP-related, NAD-associated energy failure, and rescue with TCA metabolites.     

Protective roles of cytoplasmic p21Cip1 /Waf1 in senolysis and ferroptosis of lung cancer cells

ObjectiveTherapy‐induced senescent cancer cells increase the expression of the cyclin‐dependent kinase inhibitors p16^Ink4a and p21^Cip1/Waf1. Given that p21 regulates not only the cell cycle but also cell death, we investigated the roles of p21 in cell death using a p16‐negative A549 human lung adenocarcinoma cell line.MethodsSenescence was induced by doxorubicin (DXR) or pemetrexed (PEM). The protein expression of p21 was examined by immunoblot. Cell death, reactive oxygen species (ROS) and lipid peroxidation were determined by flow cytometry. ABT‐263 and ABT‐737 were used as senolytic drugs. In vivo growth of A549 cells with different levels of p21 and their sensitivity to PEM were examined in xenograft models.ResultsDXR‐induced senescent A549 cells increased the expression of cytoplasmic p21, and the sensitivity to ABT‐263 was augmented in p21‐knockout A549 (A549‐KOp21) cells. A similar senolytic effect was observed when PEM was combined with ABT‐737. PEM alone induced a higher level of non‐apoptotic cell death, ferroptosis, in A549‐KOp21 cells than in A549 cells. Although there was no difference in the level of lipid peroxidation, ROS levels were higher in PEM‐treated A549‐KOp21 cells than in PEM‐treated A549 cells. A loss of p21 increased the sensitivity of A549 cells to PEM both in vitro and in vivo. A clinical database analysis showed that CDKN1A ^high lung adenocarcinoma patients had a poorer prognosis compared to CDKN1A ^low patients.ConclusionCytoplasmic p21, which was increased in therapy‐induced senescent lung cancer cells, plays protective roles in senolysis and ferroptosis.

Doxorubicin or pemetrexed could induce senescence in p21‐expressing parental A549 cells and increase the expression of cytoplasmic p21. However, such drug‐induced senescent A549 cells were relatively resistant to apoptosis by senolytic drugs. By contrast, p21‐knockout A549 (A549‐KOp21) cells increased their sensitivity to senolytic drugs. On the other hand, pemetrexed induced a higher level of non‐apoptotic cell death, ferroptosis, in A549‐KOp21 cells than in A549 cells. These findings highlight the protective roles of cytoplasmic p21 against senolysis and ferroptosis in therapy‐induced senescent lung cancer cells.     

Structural and Functional Analysis of Transmembrane XI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys⁴⁴⁷-Lys⁴⁷²) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp⁴⁴⁷-Tyr⁴⁵⁴ and Phe⁴⁶⁰-Lys⁴⁷¹ at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly⁴⁵⁵-Gly⁴⁵⁹. TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na⁺/H⁺ exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu⁴⁶⁵ contributing to the pore.The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1)⁴ is a ubiquitous integral membrane protein that regulates intracellular pH. It mediates removal of a single intracellular proton in exchange for an extracellular sodium ion (1). NHE1 has many functions aside from protection of cells from intracellular acidification (2). It promotes cell growth and differentiation (3), regulates sodium fluxes and cell volume after challenge by osmotic shrinkage (4), and has been demonstrated to be involved in modulating cell motility (5). In addition its activity is important in invasiveness of neoplastic breast cancer cells (6). NHE1 also plays critical roles in heart disease. It has a contributing role in heart hypertrophy and in the damage that occurs during ischemia and reperfusion. Inhibition of NHE1 with Na⁺/H⁺ exchanger inhibitors protects the myocardium during various disease states (7-10).NHE1 is composed of two general regions, an N-terminal membrane domain of ∼500 amino acids and a C-terminal regulatory domain of ∼315 amino acids (1, 8). The membrane domain is responsible for ion movement and an analysis of topology by cysteine scanning accessibility suggested it has 3 membrane-associated segments and 12 integral transmembrane segments (11) (Fig. 1A). The mechanism of transport of the membrane domain is of great interest both from a scientific viewpoint and in the design of improved NHE1 inhibitors that may be necessary for clinical use (1). In this regard, we have recently characterized the functionally important residues and the structure of both TM IV and TM VII. Prolines 167 and 168 of TM IV were critical to NHE1 function (12) and cysteine-scanning mutagenesis was used to show that Phe¹⁶¹ is a pore lining residue critical to transport. Analysis of the structure of TM IV showed that TM IV is composed of one region of β-turns, an extended middle region including Pro¹⁶⁷-Pro¹⁶⁸, and a helical region (13). TM VII was much more typical of a transmembrane helix although it was interrupted with a break in the helix at the functionally critical residues Gly²⁶¹-Glu²⁶² (14).Open in a separate windowFIGURE 1.Models of the Na⁺/H⁺ exchanger. A, simplified topological model of the transmembrane domain of the NHE1 isoform of the Na⁺/H⁺ exchanger as described earlier (11). EL, extracellular loop; IL, intracellular loop. B, model of amino acids present in TM XI.Another important TM segment of the Na⁺/H⁺ exchanger is TM XI (Fig. 1B). Several different lines of evidence have suggested that it is critical to NHE1 function. A recent study generated chimeras of NHE1 from various species and found that a region including TM XI was important in determining NHE1 inhibitor sensitivity (15). More specifically, mutagenesis of several amino acids of TM XI has shown that it is likely involved in either ion transport or proper targeting to the plasma membrane. Two mutants in TM XI, Y454C and R458C, are retained in the endoplasmic reticulum (16). In addition, mutation of Gly⁴⁵⁵ and Gly⁴⁵⁶ in TM XI shift the pH_i dependence of the exchanger to the alkaline side, whereas mutation of Arg⁴⁴⁰ in intracellular loop 5 at the N-terminal end of TM XI shifts the pH_i dependence to make it more acidic (17, 18). Also, the structure of the bacterial Na⁺/H⁺ exchanger NhaA has been elucidated. Both TM IV and TM XI play a critical role forming an assembly that cross, with each being a helix, an extended polypeptide and a short helix (19). We found that TM IV of NHE1 has a similar structure and function to that of TM IV of NhaA (2, 13), leaving open the possibility that TM XI of NHE1 is also similar in structure and function to TM XI of NhaA.For these reasons, we undertook a systematic examination of the structural and functional aspects of TM XI of the NHE1 isoform of the Na⁺/H⁺ exchanger. The sequence of human TM XI of NHE1 is ⁴⁴⁹QFIIAYGGLRGAIAFSLGYLLD⁴⁷⁰. In this study we use cysteine scanning mutagenesis and site-specific mutagenesis to identify and characterize critical pore lining residues of the protein. We also use nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of a synthetic peptide representing TM XI in dodecylphosphocholine (DPC) micelles. Evidence has suggested that TM segments of membrane proteins possess all the structural information required to form their higher order structures in their amino acid sequence (20). This has been demonstrated in earlier studies on membrane protein segments such as the cystic fibrosis transmembrane conductance regulator (21), a fungal G-protein-coupled receptor (22), bacteriorhodopsin (23, 24), and rhodopsin (25), where it was shown that isolated TM segments from membrane proteins had structures in good agreement with the segments of the entire protein. Also, the use of DPC micelles has been shown to be an excellent membrane mimetic environment for these studies (26, 27). Our study identifies Leu⁴⁶⁵ as contributing to the pore of the protein and shows that the structure of TM XI consists of two helices corresponding to Asp⁴⁴⁷-Tyr⁴⁵⁴ and Phe⁴⁶⁰-Lys⁴⁷¹ at the N and C termini, respectively, connected by a flexible region at residues 455-459. The structure of TM XI was similar to the x-ray structure of TM XI of NhaA.     

Unique backbone-water interaction detected in sphingomyelin bilayers with 1H/31P and 1H/13C HETCOR MAS NMR spectroscopy

Two-dimensional ¹H/³¹P dipolar heteronuclear correlation (HETCOR) magic-angle spinning nuclear magnetic resonance (NMR) is used to investigate the correlation of the lipid headgroup with various intra- and intermolecular proton environments. Cross-polarization NMR techniques involving ³¹P have not been previously pursued to a great extent in lipid bilayers due to the long ¹H-³¹P distances and high degree of headgroup mobility that averages the dipolar coupling in the liquid crystalline phase. The results presented herein show that this approach is very promising and yields information not readily available with other experimental methods. Of particular interest is the detection of a unique lipid backbone-water intermolecular interaction in egg sphingomyelin (SM) that is not observed in lipids with glycerol backbones like phosphatidylcholines. This backbone-water interaction in SM is probed when a mixing period allowing magnetization exchange between different ¹H environments via the nuclear Overhauser effect (NOE) is included in the NMR pulse sequence. The molecular information provided by these ¹H/³¹P dipolar HETCOR experiments with NOE mixing differ from those previously obtained by conventional NOE spectroscopy and heteronuclear NOE spectroscopy NMR experiments. In addition, two-dimensional ¹H/¹³C INEPT HETCOR experiments with NOE mixing support the ¹H/³¹P dipolar HETCOR results and confirm the presence of a H₂O environment that has nonvanishing dipolar interactions with the SM backbone.     

In Vivo Tumor Growth Rate Measured by US in Preoperative Period and Long Term Disease Outcome in Breast Cancer Patients

Objective

The aim of our study was to evaluate the effect of tumor growth rate, calculated from tumor size measurements by US, on breast cancer patients’ outcome.

Patients and Methods

Breast cancer patients who received at least two serial breast ultrasonographies (US) in our institution during preoperative period and were surgically treated between 2002 and 2010 were reviewed. Tumor growth rate was determined by specific growth rate (SGR) using the two time point tumor sizes by US.

Results

A total of 957 patients were analyzed. The median duration between initial and second US was 28 days (range, 8–140). The median initial tumor size was 1.7cm (range, 0.4–7.0) and median second size was 1.9cm (range, 0.3–7.2). 523(54.6%) cases had increase in size. The median SGR(x10^-2) was 0.59 (range, -11.90~31.49) and mean tumor doubling time was 14.51 days. Tumor growth rate was higher when initial tumor size was smaller. Lymphovascular invasion, axillary lymph node metastasis, and higher histologic grade were significantly associated with higher SGR. SGR was significantly associated with disease-free survival (DFS) in a univariate analysis (p = 0.04), but not in a multivariate Cox analysis (p>0.05). High SGR was significantly associated with worse DFS in a subgroup of initial tumor size >2cm (p = 0.018), but not in those with tumor size <2cm (p>0.05).

Conclusion

Our results showed that tumor growth rate measured by US in a relatively short time interval was associated with other worse prognostic factors and DFS, but it was not an independent prognostic factor in breast cancer patients.     

Toxoplasma gondii Inhibits Covalent Modification of Histone H3 at the IL-10 Promoter in Infected Macrophages

Infection of macrophages with the protozoan parasite Toxoplasma gondii results in inhibition of a large panel of LPS-responsive cytokines, including TNF-α, while leaving others such as IL-10 intact. Recent studies provide evidence that the parasite interferes with chromatin remodeling at the TNF-α promoter that is normally associated with LPS stimulation, but that is not required for TLR4 induction of IL-10. Here, we examined the effect of Toxoplasma on IL-10 induced by simultaneous signaling through TLR4 and FcγR, a combined stimulus that triggers histone H3 covalent modification at the IL-10 promoter resulting in high level IL-10 cytokine production. We show that the parasite inhibits high level IL-10 production and prevents histone H3 Ser¹⁰ phosphorylation and Lys^9/14 acetylation at the IL-10 promoter. These results provide compelling evidence that T. gondii targets the host cell chromatin remodeling machinery to down-regulate cytokine responses in infected macrophages.     

Combined 1H and 31P NMR studies of cerebral metabolism in vivo

NMR spectroscopic methods have recently been developed for measurement of several concentrated cerebral metabolites in vivo. At present, 31P spectra from the brain permit detection of ATP, PCr, Pi, and certain sugar and lipid phosphates. The resonant frequency of Pi also provides a measure of cerebral pHi, and under some conditions ADP concentration can be calculated from information available in the 31P spectrum. The 1H spectrum of brain provides measurements of lactate, creatine, and several amino acids and choline-containing compounds. Both kinds of spectra can be obtained from the same subject. Our group at Yale used combined 31P and 1H methods to demonstrate that loss and recovery of phosphate energy stores and concomitant changes in cerebral amino acids during hypoglycemic coma in rodents could be observed in vivo. We then used the same methods to show that cerebral pHi can be normal while lactate is elevated in status epilepticus. NMR spectroscopy performed in vivo provides an array of chemically specific measurements unavailable by any other non-invasive method. It is thought to be entirely free of deleterious biological effects; hence, its potential for use in humans is considerable.     

P2Y6 receptor‐dependent microglial phagocytosis of synapses mediates synaptic and memory loss in aging

Aging causes loss of brain synapses and memory, and microglial phagocytosis of synapses may contribute to this loss. Stressed neurons can release the nucleotide UTP, which is rapidly converted into UDP, that in turn activates the P2Y₆ receptor (P2Y₆R) on the surface of microglia, inducing microglial phagocytosis of neurons. However, whether the activation of P2Y₆R affects microglial phagocytosis of synapses is unknown. We show here that inactivation of P2Y₆R decreases microglial phagocytosis of isolated synapses (synaptosomes) and synaptic loss in neuronal–glial co‐cultures. In vivo, wild‐type mice aged from 4 to 17 months exhibited reduced synaptic density in cortical and hippocampal regions, which correlated with increased internalization of synaptic material within microglia. However, this aging‐induced synaptic loss and internalization were absent in P2Y₆R knockout mice, and these mice also lacked any aging‐induced memory loss. Thus, P2Y₆R appears to mediate aging‐induced loss of synapses and memory by increasing microglial phagocytosis of synapses. Consequently, blocking P2Y₆R has the potential to prevent age‐associated memory impairment.     

Balanced Production of Ribosome Components Is Required for Proper G1/S Transition in Saccharomyces cerevisiae

Cell cycle regulation is a very accurate process that ensures cell viability and the genomic integrity of daughter cells. A fundamental part of this regulation consists in the arrest of the cycle at particular points to ensure the completion of a previous event, to repair cellular damage, or to avoid progression in potentially risky situations. In this work, we demonstrate that a reduction in nucleotide levels or the depletion of RNA polymerase I or III subunits generates a cell cycle delay at the G₁/S transition in Saccharomyces cerevisiae. This delay is concomitant with an imbalance between ribosomal RNAs and proteins which, among others, provokes an accumulation of free ribosomal protein L5. Consistently with a direct impact of free L5 on the G₁/S transition, rrs1 mutants, which weaken the assembly of L5 and L11 on pre-60S ribosomal particles, enhance both the G₁/S delay and the accumulation of free ribosomal protein L5. We propose the existence of a surveillance mechanism that couples the balanced production of yeast ribosomal components and cell cycle progression through the accumulation of free ribosomal proteins. This regulatory pathway resembles the p53-dependent nucleolar-stress checkpoint response described in human cells, which indicates that this is a general control strategy extended throughout eukaryotes.     

ESR1 Amplification in Breast Cancer by Optimized RNase FISH: Frequent but Low-Level and Heterogeneous

Prevalence of ESR1 amplification in breast cancer is highly disputed and discrepancies have been related to different technical protocols and different scoring approaches. In addition, pre-mRNA artifacts have been proposed to influence outcome of ESR1 FISH analysis. We analyzed ESR1 gene copy number status combining an improved RNase FISH protocol with multiplex ligation-dependent probe amplification (MLPA) after laser microdissection. FISH showed a high prevalence of ESR1 gains and amplifications despite RNase treatment but MLPA did not confirm ESR1 copy number increases detected by FISH in more than half of cases. We suggest that the combination of the ESR1-specific intra-tumor heterogeneity and low-level copy number increase accounts for these discrepancies.     

Fv1 Restriction and Retrovirus Vaccine Immunity in Apobec3-Deficient 129P2 Mice

Understanding the host genetics of the immune response in retrovirus infection models could provide insights for basic HIV vaccine discovery. In Friend retrovirus (FV) infection of mice, Fv1 differentially inhibits N-tropic versus B-tropic FV infection by mediating a capsid-dependent post-entry block, Fv2 susceptibility governs splenomegaly induction, and Rfv3 resistance primes a stronger neutralizing antibody response due to more potent Apobec3 activity. Apobec3 polymorphisms in inbred mouse strains correlate with Rfv3 resistance and susceptibility, with one unresolved exception. The 129/OlaHsd (129P2) mouse strain is Fv2 and Rfv3 susceptible based on genotyping, but infection of 129P2 mice with B-tropic FV resulted in strong neutralizing antibody responses and no splenomegaly. Here we confirm that 129P2 mice are Fv1^nr/nr, explaining its resistance to B-tropic FV. Infection of 129P2 mice with NB-tropic FV, which can efficiently infect mice independent of Fv1 genotype, resulted in severe splenomegaly, high levels of viremia and weak neutralizing antibody responses regardless of Apobec3 status. Notably, high-dose B-tropic FV infection of 129P2 Apobec3-deficient mice induced significant adaptive immune responses and conferred high levels of protection following challenge with pathogenic NB-tropic FV. This immunological protection complemented previous studies that N-tropic FV can act as a live-attenuated vaccine in Fv1^b/b mice. Altogether, the results obtained in 129P2 mice strengthen the conclusion that Rfv3 is encoded by Apobec3, and highlight Fv1 incompatibility as a retroviral vaccine paradigm in mice. Due to its susceptibility to disease that allows for pathogenic challenge studies, B-tropic FV infection of 129P2 mice may be a useful model to study the immunological pathways induced by retroviral capsid restriction.     

Composition of adipose tissue and marrow fat in humans by 1H NMR at 7 Tesla

Proton NMR spectroscopy at 7 Tesla (7T) was evaluated as a new method to quantify human fat composition noninvasively. In validation experiments, the composition of a known mixture of triolein, tristearin, and trilinolein agreed well with measurements by (1)H NMR spectroscopy. Triglycerides in calf subcutaneous tissue and tibial bone marrow were examined in 20 healthy subjects by (1)H spectroscopy. Ten well-resolved proton resonances from triglycerides were detected using stimulated echo acquisition mode sequence and small voxel ( approximately 0.1 ml), and T(1) and T(2) were measured. Triglyceride composition was not different between calf subcutaneous adipose tissue and tibial marrow for a given subject, and its variation among subjects, as a result of diet and genetic differences, fell in a narrow range. After correction for differential relaxation effects, the marrow fat composition was 29.1 +/- 3.5% saturated, 46.4 +/- 4.8% monounsaturated, and 24.5 +/- 3.1% diunsaturated, compared with adipose fat composition, 27.1 +/- 4.2% saturated, 49.6 +/- 5.7% monounsaturated, and 23.4 +/- 3.9% diunsaturated. Proton spectroscopy at 7T offers a simple, fast, noninvasive, and painless method for obtaining detailed information about lipid composition in humans, and the sensitivity and resolution of the method may facilitate longitudinal monitoring of changes in lipid composition in response to diet, exercise, and disease.