Showing papers by "Torrey Pines Institute for Molecular Studies published in 2006"

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

Abstract: Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.

Nature of nonfunctional envelope proteins on the surface of human immunodeficiency virus type 1.

Abstract: Human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies are thought be distinguished from nonneutralizing antibodies by their ability to recognize functional gp120/gp41 envelope glycoprotein (Env) trimers. The antibody responses induced by natural HIV-1 infection or by vaccine candidates tested to date consist largely of nonneutralizing antibodies. One might have expected a more vigorous neutralizing response, particularly against virus particles that bear functional trimers. The recent surprising observation that nonneutralizing antibodies can specifically capture HIV-1 may provide a clue relating to this paradox. Specifically, it was suggested that forms of Env, to which nonneutralizing antibodies can bind, exist on virus surfaces. Here, we present evidence that HIV-1 particles bear nonfunctional gp120/gp41 monomers and gp120-depleted gp41 stumps. Using a native electrophoresis band shift assay, we show that antibody-trimer binding predicts neutralization and that the nonfunctional forms of Env may account for virus capture by nonneutralizing antibodies. We hypothesize that these nonfunctional forms of Env on particle surfaces serve to divert the antibody response, helping the virus to evade neutralization.

A FAAH-regulated class of N-acyl taurines that activates TRP ion channels.

Abstract: Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that catabolizes several bioactive lipids in vivo. Most of the physiological substrates of FAAH characterized to date belong to the N-acyl ethanolamine (NAE) class of fatty acid amides, including the endocannabinoid anandamide, the anti-inflammatory lipid N-palmitoyl ethanolamine, and the satiating factor N-oleoyl ethanolamine. We recently identified a second structural class of fatty acid amides regulated by FAAH in vivo: the N-acyl taurines (NATs). Global metabolite profiling revealed high concentrations of long chain (≥C20) saturated NATs in the central nervous system (CNS) of FAAH(−/−) mice. Here, we use metabolite profiling to characterize the FAAH−NAT system in peripheral mouse tissues. Livers and kidneys of FAAH(−/−) mice possessed dramatic elevations in NATs, which, in contrast to those detected in the CNS, were enriched in polyunsaturated acyl chains (e.g., C20:4, C22:6). Peripheral NATs rose more than 10-fold within 1 h following ...

Regulation of Immunity by a Novel Population of Qa-1-Restricted CD8αα+TCRαβ+ T Cells

Abstract: Regulatory mechanisms involving CD8 + T cells (CD8 regulatory T cells (Tregs)) are important in the maintenance of immune homeostasis. However, the inability to generate functional CD8 Treg clones with defined Ag specificity has precluded a direct demonstration of CD8 Treg-mediated regulation. In the present study, we describe the isolation of functional lines and clones representing a novel population of TCRαβ + Tregs that control activated Vβ8.2 + CD4 T cells mediating experimental autoimmune encephalomyelitis. They express exclusively the CD8αα homodimer and recognize a peptide from a conserved region of the TCR Vβ8.2 chain in the context of the Qa-1a (CD8αα Tregs). They secrete type 1 cytokines but not IL-2. CD8αα Tregs kill activated Vβ8.2 + but not Vβ8.2 − or naive T cells. The CD8αα Tregs prevent autoimmunity upon adoptive transfer or following in vivo activation. These findings reveal an important negative feedback regulatory mechanism targeting activated T cells and have implications in the development of therapeutic strategies for autoimmune diseases and transplantation.

Effect of impact on chondrocyte viability during insertion of human osteochondral grafts.

Abstract: Background: Osteochondral grafts, used to treat chondral and osteochondral defects, require high insertional forces that may affect the viability of chondrocytes in the graft. The objectives of this study were to (1) measure the loading impact during insertion of osteochondral grafts, (2) evaluate the effect of insertional loading on chondrocyte viability, and (3) assess this effect on chondrocyte apoptosis and activation of caspase-3. Methods: The distal parts of twelve fresh femora from six adult human cadavers were harvested within seventy-two hours after the death of the donor. From each femur, four 15-mm-diameter cylindrical osteochondral grafts were isolated; two of these grafts (a total of twenty-four grafts in the study) were transplanted with standard impact insertion into recipient sockets in the other condyle of the ipsilateral femur. The other two grafts served as unloaded controls. Loads were measured during the insertion of ten of the twenty-four transplanted grafts. Full-thickness cartilage disks were then removed from the grafts, incubated for up to forty-eight hours, and analyzed for cell viability, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive reactivity, and caspase-3 activation, each as a function of the depth from the articular surface. Results: The insertion of an osteochondral graft was characterized, on the average (and standard deviation), by 10 ± 4 impacts, each generating 2.4 ± 0.9 kN of load and 13.3 ± 4.9 MPa of stress for a duration of 0.57 ± 0.13 ms with a 0.62 ± 0.25 N·s impulse. Impact insertion increased cell death in the superficial 500 μm to 21% at one hour (p < 0.001) and 47% at forty-eight hours (p < 0.001) and also increased cell death in deeper layers at forty-eight hours. Some cell death was due to apoptosis, as indicated by an increase in caspase-3 activation at eight hours (p < 0.01) and TUNEL-positive cells at forty-eight hours (p < 0.05) in the superficial 500 μm of impacted cartilage. Conclusions: Impact insertion of osteochondral grafts generates damaging loads that cause chondrocyte death, particularly in the superficial zone, mainly as a result of apoptosis mediated by the activation of caspases. Clinical Relevance: Chondrocyte death that occurs during impact insertion of osteochondral grafts may lead to compromised function. Understanding the mechanisms and consequences of such impact loading may provide insights into potential therapeutic interventions, or lead to changes in the insertion technique, to decrease the cell injury associated with impact loading.

Antibody Responses Elicited in Macaques Immunized with Human Immunodeficiency Virus Type 1 (HIV-1) SF162-Derived gp140 Envelope Immunogens: Comparison with Those Elicited during Homologous Simian/Human Immunodeficiency Virus SHIVSF162P4 and Heterologous HIV-1 Infection

Abstract: The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIV(SF162P4), and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, DeltaV2gp140 (lacking the crown of the V2 loop), DeltaV3gp140 (lacking the crown of the V3 loop), and DeltaV2DeltaV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and DeltaV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and DeltaV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by DeltaV3gp140 or DeltaV2DeltaV3gp140. In contrast, the SHIV(SF162P4)-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.

Inhibition of Fructose 1,6-Bisphosphatase Reduces Excessive Endogenous Glucose Production and Attenuates Hyperglycemia in Zucker Diabetic Fatty Rats

Abstract: Gluconeogenesis is increased in type 2 diabetes and contributes significantly to fasting and postprandial hyperglycemia. We recently reported the discovery of the first potent and selective inhibitors of fructose 1,6-bisphosphatase (FBPase), a rate-controlling enzyme of gluconeogenesis. Herein we describe acute and chronic effects of the lead inhibitor, MB06322 (CS-917), in rodent models of type 2 diabetes. In fasting male ZDF rats with overt diabetes, a single dose of MB06322 inhibited gluconeogenesis by 70% and overall endogenous glucose production by 46%, leading to a reduction in blood glucose of >200 mg/dl. Chronic treatment of freely feeding 6-week-old male Zucker diabetic fatty (ZDF) rats delayed the development of hyperglycemia and preserved pancreatic function. Elevation of lactate (∼1.5-fold) occurred after 4 weeks of treatment, as did the apparent shunting of precursors into triglycerides. Profound glucose lowering (∼44%) and similar metabolic ramifications were associated with 2-week intervention therapy of 10-week-old male ZDF rats. In high-fat diet–fed female ZDF rats, MB06322 treatment for 2 weeks fully attenuated hyperglycemia without evidence of metabolic perturbation other than a modest reduction in glycogen stores (∼20%). The studies confirm that excessive gluconeogenesis plays an integral role in the pathophysiology of type 2 diabetes and suggest that FBPase inhibitors may provide a future treatment option.

Characterizing anti-HIV monoclonal antibodies and immune sera by defining the mechanism of neutralization.

Abstract: Understanding the nature of neutralization may provide information for crafting improvements in HIV vaccines. Using JR-FL as a prototype primary pseudovirus, we first investigated anti-HIV monoclonal antibodies (mAbs) in several neutralization formats designed to elucidate the timing of neutralization. MAb b12 was most effective before receptor binding, 2G12 neutralized effectively even after CD4 binding, and X5 and a V3 loop mAb (LE311) were inactive in a standard format but were induced by sCD4. Consistent with this latter finding, native PAGE indicated that X5 and V3 mAb binding to Envelope trimers was dependent on sCD4 binding. In contrast, 2F5 and 4E10 were active even post-CD4/CCR5 engagement. We next analyzed the neutralization mechanism of a panel of HIV+ donor plasmas of various potencies. All mediated high levels of post-CD4 neutralization that was not associated with activity in the standard format. None, however, neutralized effectively in the post-CD4/CCR5 format, suggesting that 2F5/4E10-like Abs were absent or at low concentrations. Finally, we analyzed a non-neutralizing plasma spiked with mAbs b12, 2G12 or 2F5, which resulted in increases in neutralization titers consistent with the activities of the mAbs. We conclude that these methods, together with other mapping approaches, may provide a better understanding of neutralization that could be useful in vaccine research.

Redundancy in Antigen-Presenting Function of the HLA-DR and -DQ Molecules in the Multiple Sclerosis-Associated HLA-DR2 Haplotype

Abstract: The three HLA class II alleles of the DR2 haplotype, DRB1*1501, DRB5*0101, and DQB1*0602, are in strong linkage disequilibrium and confer most of the genetic risk to multiple sclerosis Functional redundancy in Ag presentation by these class II molecules would allow recognition by a single TCR of identical peptides with the different restriction elements, facilitating T cell activation and providing one explanation how a disease-associated HLA haplotype could be linked to a CD4+ T cell-mediated autoimmune disease Using combinatorial peptide libraries and B cell lines expressing single HLA-DR/DQ molecules, we show that two of five in vivo-expanded and likely disease-relevant, cross-reactive cerebrospinal fluid-infiltrating T cell clones use multiple disease-associated HLA class II molecules as restriction elements One of these T cell clones recognizes >30 identical foreign and human peptides using all DR and DQ molecules of the multiple sclerosis-associated DR2 haplotype A T cell signaling machinery tuned for efficient responses to weak ligands together with structural features of the TCR-HLA/peptide complex result in this promiscuous HLA class II restriction

Synthesis and characterization of a novel liver-targeted prodrug of cytosine-1-beta-D-arabinofuranoside monophosphate for the treatment of hepatocellular carcinoma.

Abstract: Cytotoxic nucleosides have proven to be ineffective for the treatment of hepatocellular carcinoma (HCC) due, in part, to their inadequate conversion to their active nucleoside triphosphates (NTP) in the liver tumor and high conversion in other tissues. These characteristics lead to poor efficacy, high toxicity, and a drug class associated with an unacceptable therapeutic index. Cyclic 1-aryl-1,3-propanyl phosphate prodrugs selectively release the monophosphate of a nucleoside (NMP) into CYP3A4-expressing cells, such as hepatocytes, while leaving the prodrug intact in plasma and extrahepatic tissues. This prodrug strategy was applied to the monophosphate of the well-known cytotoxic nucleoside cytosine-1-beta-D-arabinofuranoside (cytarabine, araC). Compound 19S (MB07133), in mice, achieves good liver targeting compared to araC, generating >19-fold higher cytarabine triphosphate (araCTP) levels in the liver than levels of araC in the plasma and >12-fold higher araCTP levels in the liver than in the bone marrow, representing a >120-fold and >28-fold improvement, respectively, over araC administration.

Amplification and cloning of single dna molecules using rolling circle amplification

Abstract: The present invention relates, e.g., to a method for amplifying a small number of copies (e.g. a single copy) of a single-stranded circular DNA molecule (e.g. having a size of about 5-6 kb) by an isothermal rolling circle mechanism, using random or partially random primers and a F29-type DNA polymerase. The method, which can also be used for amplifying DNAs by non-rolling types of multiple displacement amplification, comprises incubating the reaction components in a small volume, e.g. about 10 μl or less, such as about 0.6 μl or less. The degree of amplification can be about 109 fold, or higher. A method for cell-free cloning of DNA, using the rolling circle amplification method of the invention, is described.

Role of Doa1 in the Saccharomyces cerevisiae DNA Damage Response

Abstract: The cellular response to DNA damage requires not only direct repair of the damage but also changes in the DNA replication machinery, chromatin, and transcription that facilitate survival. Here, we describe Saccharomyces cerevisiae Doa1, which helps to control the damage response by channeling ubiquitin from the proteosomal degradation pathway into pathways that mediate altered DNA replication and chromatin modification. DOA1 interacts with genes involved in PCNA ubiquitination, including RAD6, RAD18, RAD5, UBC13, and MMS2, as well as genes involved in histone H2B ubiquitination or deubiquitination, including RAD6, BRE1, LGE1, CDC73, UBP8, UBP10, and HTB2. In the absence of DOA1, damage-induced ubiquitination of PCNA does not occur. In addition, the level of ubiquitinated H2B is decreased under normal conditions and completely absent in the presence of DNA damage. In the case of PCNA, the defect associated with the doa1Δ mutant is alleviated by overexpression of ubiquitin, but in the case of H2B, it is not. The data suggest that Doa1 is the major source of ubiquitin for the DNA damage response and that Doa1 also plays an additional essential and more specific role in the monoubiquitination of histone H2B.

Fatty acid-specific fluorescent probes and their use in resolving mixtures of unbound free fatty acids in equilibrium with albumin

Abstract: We report the first measurements for profiling mixtures of unbound free fatty acids. Measurements utilized fluorescent probes with distinctly different response profiles for different free fatty acids (FFA). These probes were constructed by labeling site-specific mutants of the rat intestinal fatty acid binding protein (rI-FABP) with acrylodan. The probes were produced and screened by high-throughput methods, and from more than 30 000 such probes we selected six that together have sufficient specificity and sensitivity for resolving the profile of unbound FFA (FFAu) in mixtures of different FFAu. We developed analytical methods to determine the FFAu profile from the fluorescence (ratio) response of the different probes and used these methods to determine FFAu profiles for mixtures of arachidonate, linoleate, oleate, palmitate, and stearate in equilibrium with bovine serum albumin (BSA). Measurements were performed using mixtures with a range of total FFAu concentrations, including 0.9 nM, which is similar to normal plasma levels. We also measured single FFA binding isotherms for BSA and found that binding was described well by six to seven sites with the same binding constants (Kd). The Kd values for the FFA (4-38 nM) were inversely related to the aqueous solubility of the FFA. We constructed a model with these parameters to predict the FFAu profile in equilibrium with BSA and found excellent agreement between the profiles measured using the FFA probes and those calculated with this model. These results should lead to a better understanding of albumin's role in buffering FFAu and to profiling FFAu in intra- and extracellular biological fluids.

Bis-aromatic amides and their uses as sweet flavor modifiers, tastants and taste enhancers

Abstract: The inventions disclosed herein relate to man-made bi-aromatic amide compounds that, when contacted with comestible food or drinks or pharmaceutical compositions at concentrations preferably on the order of about 100 ppm or lower, serve as sweet taste modifiers, sweet flavoring agents, or sweet flavor enhancers, for use in foods, beverages, and other comestible products, or orally administered medicinal products or compositions, optionally in the presence of or in mixtures with conventional flavoring agents such as known natural saccharide sweeteners and previously known artificial sweeteners.

System, method and kit for processing a magnified image of biological material to identify components of a biological object

Abstract: A system, method and kit for processing an original image of biological material to identify certain components of a biological object by locating the biological object in the image, enhancing the image by sharpening components of interest in the object, and applying a contour-finding function to the enhanced image to create a contour mask. The contour mask may be processed to yield a segmented image divided by structural units of the biological material.

Identification of a candidate regulatory element within the 5' flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A.

Abstract: The Igh locus is controlled by cis -acting elements, including Eμ and the 3′ IgH regulatory region which flank the C region genes within the well-studied 3′ part of the locus. Although the presence of additional control elements has been postulated to regulate rearrangements of the V H gene array that extends to the 5′ end of the locus, the 5′ border of Igh and its flanking region have not been characterized. To facilitate the analysis of this unexplored region and to identify potential novel control elements, we physically mapped the most D-distal V H segments and scanned 46 kb of the immediate 5′ flanking region for DNase I hypersensitive sites. Our studies revealed a cluster of hypersensitive sites 30 kb upstream of the most 5′ V H gene. Detection of one site, HS1, is restricted to pro-B cell lines and HS1 is accessible to restriction enzyme digestion exclusively in normal pro-B cells, the stage defined by actively rearranging Igh-V loci. Sequence motifs within HS1 for PU.1, Pax5, and E2A bind these proteins in vitro and these factors are recruited to HS1 sequence only in pro-B cells. Transient transfection assays indicate that the Pax5 binding site is required for the repression of transcriptional activity of HS1-containing constructs. Thus, our characterization of the region 5′ of the V H gene cluster demonstrated the presence of a single cluster of DNase I hypersensitive sites within the 5′ flanking region, and identified a candidate Igh regulatory region defined by pro-B cell-specific hypersensitivity and interaction with factors implicated in regulating V(D)J recombination.

Decreased specific CD8+ T cell cross‐reactivity of antigen recognition following vaccination with Melan‐A peptide

Abstract: The aim of T cell vaccines is the expansion of antigen-specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine-induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross-reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross-reactive potential of vaccine-induced cells, we analyzed the reactivity of CD8+ T cells following vaccination of HLA-A2+ melanoma patients with Melan-A peptide, incomplete Freund's adjuvant and CpG-oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan-A-reactive CD8+ T cells in vivo. A collection of predicted Melan-A cross-reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan-A-reactive CD8+ T cell clones. While Melan-A-reactive CD8+ T cells prior to vaccination are usually constituted of widely cross-reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor-reactive.

Structure-based design of a FAAH variant that discriminates between the N-acyl ethanolamine and taurine families of signaling lipids.

Abstract: Fatty acid amide hydrolase (FAAH) inactivates a large and diverse class of endogenous signaling lipids termed fatty acid amides. Representative fatty acid amides include the N-acyl ethanolamines (NAEs) anandamide, which serves as an endogenous ligand for cannabinoid receptors, and N-oleoyl and N-palmitoyl ethanolamine, which produce satiety and anti-inflammatory effects, respectively. Global metabolite profiling studies of FAAH (−/−) mice have recently identified a second class of endogenous FAAH substrates: the N-acyl taurines (NATs). To determine the metabolic and signaling functions performed by NAEs and NATs in vivo, a FAAH variant that discriminates between these two substrate classes would be of value. Here, we report the structure-guided design of a point mutant in the active site of FAAH that selectively disrupts interactions with NATs. This glycine-to-aspartate (G268D) mutant was found to exhibit wild-type kinetic parameters with NAEs, but more than a 100-fold reduction in activity with NATs att...