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Radioimmunoconjugates targeting human epidermal growth factor receptor 2 (HER2) have shown potential to noninvasively visualize HER2-positive tumors. However, the stochastic approach that has been traditionally used to radiolabel these antibodies yields poorly defined and heterogeneous products with suboptimal in vivo performance. Here, we describe a first-in-human PET study on patients with HER2-positive breast cancer evaluating the safety, biodistribution, and dosimetry of 89 Zr-site-specific (ss)-pertuzumab PET, a site-specifically labeled radioimmunoconjugate designed to circumvent the limitations of random stochastic lysine labeling. Methods: Six patients with HER2-positive metastatic breast cancer were enrolled in a prospective clinical trial. Pertuzumab was site-specifically modified with desferrioxamine (DFO) via a novel chemoenzymatic strategy and subsequently labeled with 89 Zr. Patients were administered 74 MBq of 89 Zr-ss-pertuzumab in 20 mg of total antibody intravenously and underwent PET/CT at 1 d, 3-4 d, and 5-8 d after injection. PET imaging, whole-body probe counts, and blood draws were performed to assess the pharmacokinetics, biodistribution, and dosimetry. Results: 89 Zr-ss-pertuzumab PET/CT was used to assess HER2 status and heterogeneity to guide biopsy and decide the next line of treatment at progression. The radioimmunoconjugate was able to detect known sites of malignancy, suggesting that these tumor lesions were HER2-positive. The optimal imaging time point was 5-8 d after administration, and no toxicities were observed. Dosimetry estimates from OLINDA showed that the organs receiving the highest doses (mean ± SD) were kidney (1.8 ± 0.5 mGy/MBq), liver (1.7 ± 0.3 mGy/MBq), and heart wall (1.2 ± 0.1 mGy/MBq). The average effective dose for 89 Zr-ss-pertuzumab was 0.54 ± 0.03 mSv/MBq, which was comparable to both stochastically lysine-labeled 89 Zr-DFO-pertuzumab and 89 Zr-DFO-trastuzumab. One patient underwent PET/CT with both 89 Zr-ss-pertuzumab and 89 Zr-DFO-pertuzumab 1 mo apart, with 89 Zr-ss-pertuzumab demonstrating improved lesion detection and higher tracer avidity. Conclusion: This study demonstrated the safety, dosimetry, and potential clinical applications of 89 Zr-ss-pertuzumab PET/CT. 89 Zr-ss-pertuzumab may detect more lesions than 89 Zr-DFO-pertuzumab. Potential clinical applications include real-time evaluation of HER2 status to guide biopsy and assist in treatment decisions., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Antibody-based PET tracers are exceptionally well-suited for determination of the in vivo biodistribution and quantification of therapeutic antibodies. The continued expansion in antibody-based therapeutics has accordingly driven the development towards more robust conjugation strategies in order to reliably predict the performance of such agents. We therefore aimed to evaluate the effect of site-specific labeling by enzymatic remodeling on the stability, immuno-reactivity and tumor-targeting properties of the monoclonal antibody (mAb) trastuzumab and compare it to conventional, random labeling in a HER2-positive xenograft mouse model. Methods : Trastuzumab was conjugated with the p -SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator randomly on lysine residues or site-specifically on enzymatically modified glycans using either β-galactosidase or endoglycosidase S2 prior to 89 Zr radiolabeling. 89 Zr-DFO-trastuzumab was injected into SK-OV-3 tumor-bearing NMRI nude mice. The antibody dose was titrated with either 100 µg or 500 µg of unlabeled trastuzumab. Mice underwent small animal PET/CT imaging 24, 70 and 120 hours post-injection for longitudinal assessment. Parallel experiments were conducted with an isotype control matched antibody. In vivo imaging was supported by conventional ex vivo biodistribution and HER2 immuno-histochemistry. Furthermore, site-specifically labeled 89 Zr-DFO-trastuzumab was evaluated in a panel of subcutaneous patient-derived xenograft (PDX) models. Additionally, the affinity, in vitro stability and immuno-reactivity were assessed for all tracers. Results : Site-specific labeling significantly increased PET tumor uptake (One-way ANOVA, p<0.0001 ) at all time-points when compared to random labeling. Mean tumor uptakes were 6.7 ± 1.7, 13.9 ± 3.3 and 15.3 ± 3.8 % injected dose per gram tissue (%ID/g) at 70 hours post-injection, for random, β-galactosidase or endoglycosidase S2 labeled probes, respectively. Co-injection with unlabeled trastuzumab increased the circulation time of tracers but did not alter tumor uptake notably. Site-specific probes presented with a superior in vitro stability and immuno-reactivity compared to the randomly labeled probe. Ex vivo biodistribution confirmed the data obtained by in vivo PET imaging, and site-specific 89 Zr-DFO-trastuzumab successfully detected HER2-positive tumors in PDX mouse models. Conclusion : 89 Zr-DFO-trastuzumab is well-matched for specific immuno-PET imaging of HER2-positive tumors and site-specific labeling of trastuzumab by the SiteClick TM technology minimizes the impact of the DFO chelator on immuno-reactivity, stability and biodistribution. These findings support further development of site-specifically radiolabeled mAbs for immuno-PET., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The conjugation of antibodies with cytotoxic drugs can alter their in vivo pharmacokinetics. As a result, the careful assessment of the in vivo behavior, and specifically the tumor-targeting properties, of antibody-drug conjugates represents a crucial step in their development. In order to facilitate this process, we have created a methodology that facilitates the dual labeling of an antibody with both a toxin and a radionuclide for positron emission tomography (PET). To minimize the impact of these modifications, this chemoenzymatic approach leverages strain-promoted azide-alkyne click chemistry to graft both cargoes to the heavy chain glycans of the immuoglobulin's F c domain. As a proof-of-concept, a HER2-targeting trastuzumab immunoconjugate was created bearing both a monomethyl auristatin E (MMAE) toxin as well as the long-lived positron-emitting radiometal 89 Zr ( t 1/2 ≈ 3.3 days). Both the tumor targeting and therapeutic efficacy of the 89 Zr-trastuzumab-MMAE immunoconjugate were validated in vivo using a murine model of HER2-expressing breast cancer. The site-specifically dual-labeled construct enabled the clear visualization of tumor tissue via PET imaging, producing tumoral uptake of ∼70%ID/g. Furthermore, a longitudinal therapy study revealed that the immunoconjugate exerts significant antitumor activity, leading to a >90% reduction in tumor volume over the course of 20 days.

The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of strategies for the multimodal PET/NIRF imaging of cancer a very enticing prospect. Indeed, in the context of colorectal cancer, a single multimodal PET/NIRF imaging agent could be used to stage the disease, identify candidates for surgical intervention, and facilitate the image-guided resection of the disease. While antibodies have proven to be highly effective vectors for the delivery of radioisotopes and fluorophores to malignant tissues, the use of radioimmunoconjugates labeled with long-lived nuclides such as 89 Zr poses two important clinical complications: high radiation doses to the patient and the need for significant lag time between imaging and surgery. In vivo pretargeting strategies that decouple the targeting vector from the radioactivity at the time of injection have the potential to circumvent these issues by facilitating the use of positron-emitting radioisotopes with far shorter half-lives. Here, we report the synthesis, characterization, and in vivo validation of a pretargeted strategy for the multimodal PET and NIRF imaging of colorectal carcinoma. This approach is based on the rapid and bioorthogonal ligation between a trans -cyclooctene- and fluorophore-bearing immunoconjugate of the huA33 antibody (huA33-Dye800-TCO) and a 64 Cu-labeled tetrazine radioligand ( 64 Cu-Tz-SarAr). In vivo imaging experiments in mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts clearly demonstrate that this approach enables the non-invasive visualization of tumors and the image-guided resection of malignant tissue, all at only a fraction of the radiation dose created by a directly labeled radioimmunoconjugate. Additional in vivo experiments in peritoneal and patient-derived xenograft models of colorectal carcinoma reinforce the efficacy of this methodology and underscore its potential as an innovative and useful clinical tool., Competing Interests: The authors have declared that no competing interest exists.

In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifically modified huA33-trans-cyclooctene immunoconjugate ((ss)huA33-PEG12-TCO). A bioconjugation strategy that harnesses enzymatic transformations and strain-promoted azide-alkyne click chemistry was used to site-specifically append PEGylated TCO moieties to the heavy chain glycans of the colorectal cancer-targeting huA33 antibody. Preclinical in vivo validation studies were performed in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts. To this end, mice were administered (ss)huA33-PEG12-TCO via tail vein injection and-following accumulation intervals of 24 or 48 h-(64)Cu-Tz-SarAr. PET imaging and biodistribution studies reveal that this strategy clearly delineates tumor tissue as early as 1 h post-injection (6.7 ± 1.7%ID/g at 1 h p.i.), producing images with excellent contrast and high tumor-to-background activity concentration ratios (tumor:muscle = 21.5 ± 5.6 at 24 h p.i.). Furthermore, dosimetric calculations illustrate that this pretargeting approach produces only a fraction of the overall effective dose (0.0214 mSv/MBq; 0.079 rem/mCi) of directly labeled radioimmunoconjugates. Ultimately, this method effectively facilitates the high contrast pretargeted PET imaging of colorectal carcinoma using a site-specifically modified immunoconjugate.

Molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF)-guided delineation of surgical margins could greatly enhance the diagnosis, staging, and resection of pancreatic cancer. PET and NIRF optical imaging offer complementary clinical applications, enabling the noninvasive whole-body imaging to localize disease and identification of tumor margins during surgery, respectively. We report the development of PET, NIRF, and dual-modal (PET/NIRF) imaging agents, using 5B1, a fully human monoclonal antibody that targets CA19.9, a well-established pancreatic cancer biomarker. Desferrioxamine (DFO) and/or a NIRF dye (FL) were conjugated to the heavy-chain glycans of 5B1, using a robust and reproducible site-specific (ss) labeling methodology to generate three constructs ((ss)DFO-5B1, (ss)FL-5B1, and (ss)dual-5B1) in which the immunoreactivity was not affected by the conjugation of either label. Each construct was evaluated in a s.c. xenograft model, using CA19.9-positive (BxPC3) and -negative (MIAPaCa-2) human pancreatic cancer cell lines. Each construct showed exceptional uptake and contrast in antigen-positive tumors with negligible nonspecific uptake in antigen-negative tumors. Additionally, the dual-modal construct was evaluated in an orthotopic murine pancreatic cancer model, using the human pancreatic cancer cell line, Suit-2. The (ss)dual-5B1 demonstrated a remarkable capacity to delineate metastases and to map the sentinel lymph nodes via tandem PET-computed tomography (PET/CT) and NIRF imaging. Fluorescence microscopy, histopathology, and autoradiography were performed on representative sections of excised tumors to visualize the distribution of the constructs within the tumors. These imaging tools have tremendous potential for further preclinical research and for clinical translation.

Protein acylation plays a critical role in protein localization and function. Acylation is essential for human immunodeficiency virus 1 (HIV-1) assembly and budding of HIV-1 from the plasma membrane in lipid raft microdomains and is mediated by myristoylation of the Gag polyprotein and the copackaging of the envelope protein is facilitated by colocalization mediated by palmitoylation. Since the viral accessory protein NEF has been shown to alter the substrate specificity of myristoyl transferases, and alter cargo trafficking lipid rafts, we hypothesized that HIV-1 infection may alter protein acylation globally. To test this hypothesis, we labeled HIV-1 infected cells with biomimetics of acyl azides, which are incorporated in a manner analogous to natural acyl-Co-A. A terminal azide group allowed us to use a copper catalyzed click chemistry to conjugate the incorporated modifications to a number of substrates to carry out SDS-PAGE, fluorescence microscopy, and enrichment for LC-MS/MS. Using LC-MS/MS, we identified 103 and 174 proteins from the myristic and palmitic azide enrichments, with 27 and 45 proteins respectively that differentiated HIV-1 infected from uninfected cells. This approach has provided us with important insights into HIV-1 biology and is widely applicable to many virological systems., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The complementary nature of positron emission tomography (PET) and optical imaging (OI) has fueled increasing interest in the development of multimodal PET/OI probes that can be employed during the diagnosis, staging, and surgical treatment of cancer. Due to their high selectivity and affinity, antibodies have emerged as promising platforms for the development of hybrid PET/OI agents. However, the lack of specificity of many bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs. To circumvent this issue, we have developed a chemoenzymatic strategy for the construction of multimodal PET/OI immunoconjugates that have been site-specifically labeled on the heavy chain glycans. The methodology consists of four steps: (1) the enzymatic removal of the terminal galactose residues on the heavy chain glycans; (2) the enzymatic incorporation of azide-bearing galactose (GalNAz) residues into the heavy chain glycans; (3) the strain-promoted click conjugation of chelator- and fluorophore-modified dibenzocyclooctynes to the azide-modified sugars; and (4) the radiolabeling of the immunoconjugate. For proof-of-concept, a model system was created using the colorectal cancer-targeting antibody huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal (89)Zr, and the near-infrared fluorescent dye Alexa Fluor 680. The bioconjugation strategy is robust and reproducible, reliably producing well-defined and immunoreactive conjugates labeled with (89)Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of the two reporters. In in vivo PET and fluorescence imaging experiments, a hybrid (89)Zr- and Alexa Fluor 680-labeled huA33 conjugate displayed high levels of specific uptake (>45% ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts.

An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal (89)Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with (89)Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing (89)Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to highly selective tumor uptake (67.5 ± 5.0%ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic.

A recently developed proteomic strategy, the "GG-azide"-labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido-geranylgeranyl analog and chemoselective derivatization of azido-geranylgeranyl-modified proteins by the "click" chemistry, using a tetramethylrhodamine-alkyne. The resulting conjugated proteins can be separated by 1-D or 2-D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC-MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The "GG-azide"-labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post-translational modifications.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the synthesis of a truncated prelamin A, commonly called progerin, that contains a carboxyl-terminal farnesyl lipid anchor. The farnesyl lipid anchor helps to target progerin to membrane surfaces at the nuclear rim, where it disrupts the integrity of the nuclear lamina and causes misshapen nuclei. Several lines of evidence have suggested that progerin's farnesyl lipid anchor is crucial for the emergence of disease phenotypes. Because a geranylgeranyl lipid is approximately 45-fold more potent than a farnesyl lipid in anchoring proteins to lipid membranes, we hypothesized that a geranylgeranylated version of progerin might be more potent in eliciting disease phenotypes. To test this hypothesis, we used gene targeting to create mice expressing geranylgeranylated progerin (Lmna(ggHG/+)). We then compared Lmna(ggHG/+) mice, side-by-side, with otherwise identical mice expressing farnesylated progerin (Lmna(HG/+)). Geranylgeranylation of progerin in Lmna(ggHG/+) cells and farnesylation of progerin in Lmna(HG/+) cells was confirmed by metabolic labeling. Contrary to our expectations, Lmna(ggHG/+) mice survived longer than Lmna(HG/+) mice. The Lmna(ggHG/+) mice also exhibited milder bone disease. The steady-state levels of progerin, relative to lamin C, were lower in Lmna(ggHG/+) mice than in Lmna(HG/+) mice, providing a potential explanation for the milder disease in Lmna(ggHG/+) mice.

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.

The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] cycloaddition between a terminal alkyne and an azide. 5-ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis, just like BrdU. While the BrdU assay requires harsh chemical or enzymatic disruption of helical DNA structure to allow for direct measurement of cells in the S-phase by the anti-BrdU antibody, the EdU method does not. Elimination of this requirement results in the preservation of helical DNA structure and other cell surface epitopes, decreased assay time, and increased reproducibility.

The Multiplexed Proteomics (MP) technology is a new approach that permits quantitative, multicolor fluorescence detection of proteins in one-dimensional or two-dimensional gels. This methodology allows for multiplexed identification and differential analysis of phosphoproteins, glycoproteins, and total proteins within a single gel electrophoresis experiment. Here the MP system was applied to the differential proteomic analysis of pregnancy-induced refractoriness to breast cancer using a rat model system. Differential analyses identified multiple proteins with altered phosphorylation, glycosylation, or protein expression patterns.

A new solid-phase, fluorescence-based protein assay was developed that quantifies proteins in the presence of detergents, urea and reducing agents (one-dimensional sodium dodecyl sulfate (1-D SDS) lysis buffers and urea isoelectric focusing (IEF) buffers). A specially designed 96-well microplate facilitates application of protein samples to the assay paper and allows easy quantitation of samples using fluorescence microplate readers (top or bottom reading format). Alternatively, stained membranes may be directly scanned using a variety of different laser or charge-coupled device (CCD)-based imaging devices with UV or visible imaging capabilities. Since protein is specifically bound to the membrane, contaminants are readily washed away, avoiding interference with the protein measurement. The protein assay has a dynamic range extending from 10 ng to 5 microg of protein per microliter and requires only 1 microL of sample, which is ideal for samples destined for electrophoresis. The protein-to-protein variability of staining of ten different proteins was determined to be comparable with that of the bicinchoninic acid (BCA) and Lowry assays (16%). Additionally, the quality of the assay according to Z-factor analyses is excellent., (Copyright 2004 Wiley-VCH Verlag GmbH and Co.)

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.

Archaeosomes, liposomes made from polar ether lipids of archaea, show promise for vaccine and drug delivery applications. The potential toxicity of intravenously (14, 70, or 140 mg/kg/day for 5 consecutive days) and orally (gavaged at 55, 275, or 550 mg/kg/day for 10 consecutive days) administered unilamellar archaeosomes, prepared from the total polar lipids (TPLs) extracted from several species of archaea, was assessed in female BALB/c mice. Liposomes prepared from an ester phospholipid composition were included for comparative purposes. Control groups of mice were administered 0.1 ml phosphate-buffered saline (PBS) by either route. Animals were monitored at least once daily for temperature, body weight, and clinical signs of adverse reactions. One day after the last dose, the mice were sacrificed. Blood was collected for selected biochemical/enzyme analyses, and the major organs (heart, lungs, liver, spleen, kidneys) were weighed and examined macroscopically. In addition, the spleens were examined histologically. At the two lower dosages of intravenously administered vesicles, there were no significant indications of toxicity, as compared with the PBS-administered control group. At the highest intravenous dose of 140 mg/kg/day, archaeosomes prepared from the TPL of the extreme halophiles, Halobacterium salinarum and Natronobacterium magadii, indicated potential toxicity, as evidenced by clinical signs (hyperactivity and/or piloerection), drop in body temperature, and loss in body weight. Spleens from mice administered some archaeosomes types, primarily at the highest intravenous dose tested, were enlarged, had increased organ weight, and microscopic examination revealed mild to moderate expansion of the red pulp with increased numbers of hematopoietic cells, but no changes in the white pulp. There were similar clinical signs at one or more of the higher oral doses of the ester liposomes and some of the archaeosome types; however, no other apparent toxicity was observed. Based on this limited mouse study, archaeosomes were generally well tolerated after intravenous or oral delivery at the dosages so indicated in this study.

The function and viability of vertebrate photoreceptors requires the daily phagocytosis of photoreceptor outer segments (OS) by the adjacent retinal pigment epithelium (RPE). We demonstrate here a critical role in this process for Gas6 and by implication one of its receptor protein tyrosine kinases (RTKs), Mertk (Mer). Gas6 specifically and selectively stimulates the phagocytosis of OS by normal cultured rat RPE cells. The magnitude of the response is dose-dependent and shows an absolute requirement for calcium. By contrast the Royal College of Surgeons (RCS) rat RPE cells, in which a mutation in the gene Mertk results in the expression of a truncated, non-functional receptor, does not respond to Gas6. These data strongly suggest that activation of Mertk by its ligand, Gas6, is the specific signaling pathway responsible for initiating the ingestion of shed OS. Moreover, photoreceptor degeneration in the RCS rat retina, which lacks Mertk, and in humans with a mutation in Mertk, strongly suggests that the Gas6/Mertk signaling pathway is essential for photoreceptor viability. We believe that this is the first demonstration of a specific function for Gas6 in the eye., (Copyright 2001 Academic Press.)

The in vitro stability of archaeosomes made from the total polar lipids of Methanosarcina mazei, Methanobacterium espanolae or Thermoplasma acidophilum, was evaluated under conditions encountered in the human gastrointestinal tract. At acidic pH, multilamellar vesicles (MLV) prepared from T. acidophilum lipids were the most stable, releasing approximately 80, 20, 10 and 5% of encapsulated 14C-sucrose at pH 1.5, 2.0, 2.5 and 6.2, respectively, after 90 min at 37 degrees C. Archaeosomes from M. mazei lipids were the least stable. For each type of total polar lipid, unilamellar vesicles (ULV) were less stable than the corresponding MLV vesicles. Pancreatic lipase had relatively minor effect on the stability of archaeosomes made from either of the three types of total polar lipids, causing the release of 12-27% of the encapsulated 5(6)-carboxyfluorescein (CF) from ULV and MLV after 90 min at 37 degrees C. In simulated human bile at pH 6.2, MLV from M. mazei total polar lipids lost 100% of the encapsulated CF after 90 min at 37 degrees C, whereas those from the polar lipids of M. espanolae or T. acidophilum lost approximately 85% of the marker. Pancreatic lipase and simulated human bile had no synergistic effect on the release of carboxyfluorescein from ULV or MLV prepared from any of the total polar lipids. After 90 min in the combined presence of these two stressors at pH 6.2, the leakage of fluorescein conjugated bovine serum albumin from MLV prepared from T. acidophilum lipids was similar to that of CF, and 13% of the initially present vesicles appeared to be intact. These results indicate that archaeosomes show stability properties indicative of potential advantages in developing applications as an oral delivery system.

The biodistribution of orally and intravenously administered archaeosomes in mice was compared to that of archaeosomes containing either coenzyme Q10 (archaeosome-CoQ10), polyethylene glycol (archaeosome-PEG), or PEG plus CoQ10 (archaeosome-PEG-CoQ10). The archaeosome formulations were prepared by a reverse-phase evaporation method using the total polar lipids from the archaeobacterium Methanosarcina mazei. In the case of oral gavage, the most striking observation was that a significantly (p < 0.05) higher concentration (42.28+/-4.17%) of administered dose was found in the stomach content 3 h after administration of unmodified archaeosomes, as compared to that of archaeosome-CoQ10 (16.98+/-2.48%) and archaeosome-PEG-CoQ10 (5.8 +/-4.05"/ vesicles. This correlated with an increased uptake, notably of the archaeosome-PEG-CoQ 0 vesicles.,into liver and spleen; however, no more than 7% of the administered dose was found in liver, spleen and blood at any time point studied. In the case of intravenous administration, a significantly higher percentage of injected dose of unmodified archaeosomes was found in the liver (66.4 +/-.92%) and spleen (11.445+/-.68%) at 48 h, compared to archaeosome-CoQ10, archaeosome-PEG, and archaeosome-PEG-CoQ10 vesicles. The combination of PEG and CoQ10 significantly prolonged the circulation of archaeosomes in the blood, but after 48 h the amount of the vesicle marker in blood had declined to only about 0.5% of administered dose. These data indicate that the biodistribution of archaeosome formulations given orally or intravenously can be altered significantly by incorporating PEG or CoQ 10, alone or in combination, and these vesicles have the potential to act as a carrier for therapeutics and vaccines.

Cultured rabbit parietal cells were used to evaluate morphological responses to activators and inhibitors of HCl secretion. Immunofluorescence was used to localize the proton pump protein, H, K-ATPase, and the apical membrane-cytoskeletal linker protein, ezrin; fluorescent-labeled phalloidin was used as a marker of F-actin. Treatment of healthy control parietal cells with secretagogues resulted in exaggerated swelling of apical membrane vacuoles, presumably with the accumulation of HCl and water. Thus stimulation-associated swelling of apical vacuoles was blocked by inhibitors that work at various steps in the secretion-activation cascade. When secretion was blocked by agents that prevent the translocation of H,K-ATPase-rich tubulovesicles to apical membrane vacuoles (such as H2-receptor antagonists and protein kinase A inhibitors), the general resting morphology was maintained. ME-3407 (a functional analogue of wortmannin) was unique in preventing H, K-ATPase redistribution and effecting the delocalization of ezrin from apical membrane vacuoles. When secretion was blocked by agents that inhibit the H+ pump or induce H+ backflux, the translocation of H,K-ATPase to apical membrane vacuoles occurred but the large vacuolar swelling associated with HCl and H2O accumulation was greatly diminished. These data support the membrane recycling/recruitment hypothesis of HCl secretion in which H, K-ATPase-rich tubulovesicles are recruited from a cytoplasmic domain to the apical surface, and they are inconsistent with models proposing that the tubulovesicles, regardless of shape, are contiguous with the apical plasma membrane. These studies also demonstrate the utility of the parietal cell culture model in distinguishing a general site of action for various inhibitors and antisecretory agents.

In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.

Actin depolymerizing factor (ADF) occurs naturally in two forms, one of which contains a phosphorylated Ser and does not bind G-actin or depolymerize F-actin. Removal of this phosphate in vitro by alkaline phosphatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [32P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one 32P-labeled peptide. Further digestion with endoproteinase Asp-N and mass spectrometric analysis showed that this peptide came from the N terminus of ADF. Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted it to a peptide of mass 673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this peptide identified the phosphorylated Ser as the encoded Ser3 (Ser2 in the processed protein). HeLa cells, transfected with either chick wild-type ADF cDNA or a cDNA mutated to code for Ala in place of Ser24 or Thr25, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo regulatory site.

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per mug of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per mug of vector DNA. However, a 380-fold improvement (3,417 transformants per mug of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.

The inhibition of methane production by Methanosaeta concilii GP6, Methanospirillum hungatei GP1, Methanobacterium espanolae GP9, and Methanobacterium bryantii M.o.H. during short-term (6-h) exposure to eight benzene ring compounds was studied. The concentration that caused 50% inhibition of the methane production rate (IC50) was dependent on the species and the toxicant. Pentachlorophenol was the most toxic of the tested compounds, with an IC50 of less than 8 mg/liter for all species except M. hungatei. Abietic acid was the next most toxic compound for all the species, with an IC50 in the range of 21.4 to 203 mg/liter. Sodium benzoate was generally the least toxic, with an IC50 in the range of 1,225 to 32,400 mg/liter. 3-Chlorobenzoate was substantially more toxic (IC50, 450 to 1,460 mg/liter) than benzoate. The inhibition by benzene, phenol, vanillic acid, and toluene was intermediate to that of pentachlorophenol and benzoate. Long-term incubation (days) studies to determine effect on growth indicated that all eight compounds were usually much more toxic than predicted from the short-term data. In these latter studies, there was generally a good correlation in the observed inhibition as determined from growth and methane production.

Bacteroides polypragmatus type strain GP4 was adapted to grow in the presence of 3.5% (w/v) ethanol by successive transfers into 1% (w/v) D-xylose media supplemented with increasing concentrations of ethanol. The maximum specific growth rate of the ethanol-adapted culture (mu = 0.30 h-1) was not affected by up to 2% (w/v) ethanol but that of the non-adapted strain declined by about 50%. The growth rate of both cultures was limited by nutrient(s) contained in yeast extract. The ethanol yield of the adapted culture (1.01 mol/mol xylose) was higher than that (0.80 mol/mol xylose) of the non-adapted strain. The adapted culture retained the ability to simultaneously ferment pentose and hexose sugars, and moreover it was not inhibited by xylose concentrations of 7-9% (w/v). This culture also readily fermented hemicellulose hydrolysates obtained by mild acid hydrolysis of either hydrogen fluoride treated or steam exploded Aspen wood. The ethanol yield from the fermentation of the hydrolysates was comparable to that obtained from xylose.

A 22-mm outside diameter screw cap test tube was modified for measuring the redox potential (Eh) of samples of anaerobic media. The screw cap was fitted with a grey butyl rubber septum with a hole through which a platinum-calomel combination electrode was inserted. The tube was shortened in length and a small side arm that could be sealed with a serum stopper and an aluminium seal was attached. This device enabled the measurement of Eh of anaerobic media without the need for continuous flushing of the vessel headspace to maintain anaerobiosis.