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Recent studies indicated that in elderly individuals, statin therapy is associated with a reduced response to influenza vaccination. The present study was designed to determine effects on the immune response to influenza vaccination induced by statin administration in a mouse model, and investigate potential approaches to improve the outcome of vaccination on the background of statin therapy. We fed middle aged BALB/c mice a high fat “western” diet (WD) alone or supplemented with atorvastatin (AT) for 14 weeks, and control mice were fed with the regular rodent diet. Mice were immunized with a single dose of subunit A/Brisbane/59/07 (H1N1) vaccine, either systemically or with dissolving microneedle patches (MNPs). We observed that a greater age-dependent decline in the hemagglutinin inhibition titers occurred in systemically-immunized mice than in MNP- immunized mice. AT dampened the antibody response in the animals vaccinated by either route of vaccine delivery. However, the MNP-vaccinated AT-treated animals had ~20 times higher total antibody levels to the influenza vaccine than the systemically vaccinated group one month postvaccination. We propose that microneedle vaccination against influenza provides an approach to ameliorate the immunosuppressive effect of statin therapy observed with systemic immunization.

Influenza virus causes life-threatening infections in pregnant women and their newborns. Immunization during pregnancy is the most effective means of preventing maternal and infant mortality/morbidity; however, influenza vaccination rates of pregnant women remain under 50%. Furthermore, the availability of vaccines in low-resource populations is limited. Skin immunization with microneedle patches (MN) is a novel and safe vaccination platform featuring thermostable vaccine formulations. Cold-chain independence and the potential for self-administration can expand influenza vaccination coverage in developing countries. In this study of pregnant BALB/c mice immunized with subunit H1N1 influenza vaccine, we demonstrate the advantage of skin vaccination over intramuscular delivery of a two-fold higher vaccine dose. MN vaccine induced superior humoral immune responses and conferred protective immunity against a lethal challenge dose of homologous influenza virus. Importantly, MN vaccination of mice at mid-gestation resulted in enhanced and long-lasting passive immunity of the offspring, measured by neutralizing antibody titers and survival rates after virus challenge. We conclude that skin vaccination using MN is a superior immunization approach with the potential to overcome immune tolerance observed in pregnancy, and lower vaccination costs through antigen dose-sparing, which is especially relevant in underserved countries.

Maternal and neonatal tetanus claim tens of thousands lives every year in developing countries, but could be prevented by hygienic practices and improved immunization of pregnant women. This study tested the hypothesis that skin vaccination can overcome the immunologically transformed state of pregnancy and enhance protective immunity to tetanus in mothers and their newborns. To achieve this goal, we developed microneedle patches (MNPs) that efficiently delivered unadjuvanted tetanus toxoid to skin of pregnant mice and demonstrated that this route induced superior immune responses in female mice conferring 100% survival to tetanus toxin challenge when compared to intramuscular vaccination. Mice born to MNP-vaccinated mothers showed detectable tetanus-specific IgG antibodies up to 12weeks of age and complete protection to tetanus toxin challenge up at 6weeks of age. In contrast, none of the 6-week old mice born to intramuscularly vaccinated mothers survived challenge. Although pregnant mice vaccinated with unadjuvanted tetanus toxoid had 30% lower IgG and IgG1 titers than mice vaccinated intramuscularly with Alum®-adjuvanted tetanus toxoid vaccine, IgG2a titers and antibody affinity maturation were similar between these groups. We conclude that skin immunization with MNPs containing unadjuvanted tetanus toxoid can confer potent protective efficacy to mothers and their offspring using a delivery method well suited for expanding vaccination coverage in developing countries.

The widely used influenza subunit vaccine would benefit from increased protection rates in vulnerable populations. Skin immunization by microneedle (MN) patch can increase vaccine immunogenicity, as well as increase vaccination coverage due to simplified administration. To further increase immunogenicity, we used granulocyte-macrophage colony stimulating factor (GM-CSF), an immunomodulatory cytokine already approved for skin cancer therapy and cancer support treatment. GM-CSF has been shown to be upregulated in skin following MN insertion. The GM-CSF-adjuvanted vaccine induced robust and long-lived antibody responses cross-reactive to homosubtypic and heterosubtypic influenza viruses. Addition of GM-CSF resulted in increased memory B cell persistence relative to groups given influenza vaccine alone and led to rapid lung viral clearance following lethal infection with homologous virus in the mouse model. Here we demonstrate that successful incorporation of the thermolabile cytokine GM-CSF into MN resulted in improved vaccine-induced protective immunity holding promise as a novel approach to improved influenza vaccination. To our knowledge, this is the first successful incorporation of a cytokine adjuvant into dissolvable MNs, thus advancing and diversifying the rapidly developing field of MN vaccination technology.

Prevention of seasonal influenza epidemics and pandemics relies on widespread vaccination coverage to induce protective immunity. In addition to a good antigenic match with the circulating viruses, the effectiveness of individual strains represented in the trivalent vaccines depends on their immunogenicity. In this study, we evaluated the immunogenicity of H1N1, H3N2, and B seasonal influenza virus vaccine strains delivered individually with a novel dissolving microneedle patch and the stability of this formulation during storage at 25 °C. Our data demonstrate that all strains retained their antigenic activity after incorporation in the dissolving patches as measured by single radial diffusion (SRID) assay and immune responses to vaccination in BALB/c mice. After a single immunization, all three antigens delivered with microneedle patches induced superior neutralizing antibody titers compared to intramuscular immunization. Cutaneous antigen delivery was especially beneficial for the less immunogenic B strain. Mice immunized with dissolving microneedle patches encapsulating influenza A/Brisbane/59/07 (H1N1) vaccine were fully protected against lethal challenge by homologous mouse-adapted influenza virus. All vaccine components retained activity during storage at room temperature for at least 3 months as measured in vitro by SRID assay and in vivo by mouse immunization studies. Our data demonstrate that dissolving microneedle patches are a promising advance for influenza cutaneous vaccination due to improved immune responses using less immunogenic influenza antigens and enhanced stability.

This paper reports on the results of a comparative study of user search logs in two large-scale, domain-specific digital libraries functioning in the United States: the National Science Digital Library and Opening History. Results demonstrate varying levels of use of advanced search options and substantial differences in the search query lengths, search query frequencies, and distribution of search categories in queries. The empirical data on how the members of the target communities search can be used in deriving important information for domain-specific digital library developers’ decisions regarding both the details of information organization and support of various search features.

Summary Background Microneedle patches provide an alternative to conventional needle-and-syringe immunisation, and potentially offer improved immunogenicity, simplicity, cost-effectiveness, acceptability, and safety. We describe safety, immunogenicity, and acceptability of the first-in-man study on single, dissolvable microneedle patch vaccination against influenza. Methods The TIV-MNP 2015 study was a randomised, partly blinded, placebo-controlled, phase 1, clinical trial at Emory University that enrolled non-pregnant, immunocompetent adults from Atlanta, GA, USA, who were aged 18–49 years, naive to the 2014–15 influenza vaccine, and did not have any significant dermatological disorders. Participants were randomly assigned (1:1:1:1) to four groups and received a single dose of inactivated influenza vaccine (fluvirin: 18 μg of haemagglutinin per H1N1 vaccine strain, 17 μg of haemagglutinin per H3N2 vaccine strain, and 15 μg of haemagglutinin per B vaccine strain) (1) by microneedle patch or (2) by intramuscular injection, or received (3) placebo by microneedle patch, all administered by an unmasked health-care worker; or received a single dose of (4) inactivated influenza vaccine by microneedle patch self-administered by study participants. A research pharmacist prepared the randomisation code using a computer-generated randomisation schedule with a block size of 4. Because of the nature of the study, participants were not masked to the type of vaccination method (ie, microneedle patch vs intramuscular injection). Primary safety outcome measures are the incidence of study product-related serious adverse events within 180 days, grade 3 solicited or unsolicited adverse events within 28 days, and solicited injection site and systemic reactogenicity on the day of study product administration through 7 days after administration, and secondary safety outcomes are new-onset chronic illnesses within 180 days and unsolicited adverse events within 28 days, all analysed by intention to treat. Secondary immunogenicity outcomes are antibody titres at day 28 and percentages of seroconversion and seroprotection, all determined by haemagglutination inhibition antibody assay. The trial is completed and registered with ClinicalTrials.gov, number NCT02438423. Findings Between June 23, 2015, and Sept 25, 2015, 100 participants were enrolled and randomly assigned to a group. There were no treatment-related serious adverse events, no treatment-related unsolicited grade 3 or higher adverse events, and no new-onset chronic illnesses. Among vaccinated groups (vaccine via health-care worker administered microneedle patch or intramuscular injection, or self-administered microneedle patch), overall incidence of solicited adverse events (n=89 vs n=73 vs n=73) and unsolicited adverse events (n=18 vs n=12 vs n=14) were similar. Reactogenicity was mild, transient, and most commonly reported as tenderness (15 [60%] of 25 participants [95% CI 39–79]) and pain (11 [44%] of 25 [24–65]) after intramuscular injection; and as tenderness (33 [66%] of 50 [51–79]), erythema (20 [40%] of 50 [26–55]), and pruritus (41 [82%] of 50 [69–91]) after vaccination by microneedle patch application. The geometric mean titres were similar at day 28 between the microneedle patch administered by a health-care worker versus the intramuscular route for the H1N1 strain (1197 [95% CI 855–1675] vs 997 [703–1415]; p=0·5), the H3N2 strain (287 [192–430] vs 223 [160–312]; p=0·4), and the B strain (126 [86–184] vs 94 [73–122]; p=0·06). Similar geometric mean titres were reported in participants who self-administered the microneedle patch (all p>0·05). The seroconversion percentages were significantly higher at day 28 after microneedle patch vaccination compared with placebo (all p 0·01). Interpretation Use of dissolvable microneedle patches for influenza vaccination was well tolerated and generated robust antibody responses. Funding National Institutes of Health.

The extracellular domain of matrix protein 2 (M2e) is conserved among influenza A viruses. The goal of this project is to develop enhanced influenza vaccines with broad protective efficacy using the M2e antigen. We designed a membrane-anchored fusion protein by replacing the hyperimmunogenic region ofSalmonella entericaserovar Typhimurium flagellin (FliC) with four repeats of M2e (4.M2e-tFliC) and fusing it to a membrane anchor from influenza virus hemagglutinin (HA). The fusion protein was incorporated into influenza virus M1-based virus-like particles (VLPs). These VLPs retained Toll-like receptor 5 (TLR5) agonist activity comparable to that of soluble FliC. Mice immunized with the VLPs by either intramuscular or intranasal immunization showed high levels of systemic M2-specific antibody responses compared to the responses to soluble 4.M2e protein. High mucosal antibody titers were also induced in intranasally immunized mice. All intranasally immunized mice survived lethal challenges with live virus, while intramuscularly immunized mice showed only partial protection, revealing better protection by the intranasal route. These results indicate that a combination of M2e antigens and TLR ligand adjuvants in VLPs has potential for development of a broadly protective influenza A virus vaccine.

This poster reports the preliminary results of a comparative transaction log analysis study of user searching at collection and item levels in a large-scale cultural heritage digital library that provides a unique option for collection-level search. Substantial changes in user searching have been observed over time, including an important trend of the growing use of collection-level search feature. Results also demonstrate differences in the search query lengths, frequencies, and distribution of search categories between the collection-level and item-level searching. The higher occurrence of place and concept categories in collection-level user searches than in item-level searches has been consistent over time. The analysis also revealed the prevalence of subject searching (including concept, object, event, place, class of persons, and ethnic group search categories) both at the collection level and at the item level. This study provides empirical data to support digital library developers' decision making regarding information organization in large-scale aggregations of digital collections.

Flotillins and caveolins represent two types of resident proteins associated with lipid rafts in mammalian cells, however, their possible cross-talk in regulating lipid raft functions remains poorly understood. In this report, we observed that siRNA-mediated down-regulation of flotillin-1 expression which disrupted lipid raft-mediated endocytosis of BODIPY FL C(5)-lactosylceramide also substantially decreased caveolin-1 level in SK-CO15 human intestinal epithelial cells. The decrease in caveolin-1 expression appeared to be specific for flotillin-1 knock-down and was not observed after down-regulation of flotillin-2. The decrease in caveolin-1 level in flotillin-1-depleted cells was not due to suppression of its mRNA synthesis and was not mimicked by cholesterol depletion of SK-CO15 cells. Furthermore, flotillin-1 dependent down-regulation of caveolin-1 was reversed after cell exposure to lysosomal inhibitor, chloroquine but not proteosomal inhibitor, MG262. Our data suggest that flotillin-1 regulates caveolin-1 level by preventing its lysosomal degradation in intestinal epithelial cells.

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of β1-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, β1-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent β1-integrin internalization. However, β1-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized β1-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased β1-integrin endocytosis. Our data suggest that, in migrating IEC, β1-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.

Chlorosomes of the photosynthetic green sulfur bacterium Chlorobium tepidum consist of bacteriochlorophyll (BChl) c aggregates that are surrounded by a lipid-protein monolayer envelope that contains ten different proteins. Chlorosomes also contain a small amount of BChl a, but the organization and location of this BChl a are not yet clearly understood. Chlorosomes were treated with sodium dodecyl sulfate (SDS), Lubrol PX, or Triton X-100, separately or in combination with 1-hexanol, and the extracted components were separated from the residual chlorosomes by ultrafiltration on centrifugal filters. When chlorosomes were treated with low concentrations of SDS, all proteins except CsmA were extracted. However, this treatment did not significantly alter the size and shape of the chlorosomes, did not extract the BChl a, and caused only minor changes in the absorption spectrum of the chlorosomes. Cross-linking studies with SDS-treated chlorosomes revealed the presence of multimers of the major chlorosome protein, CsmA, up to homooctamers. Extraction of chlorosomes with SDS and 1-hexanol solubilized all ten chlorosome envelope proteins as well as BChl a. Although the size and shape of these extracted chlorosomes did not initially differ significantly from untreated chlorosomes, the extracted chlorosomes gradually disintegrated, and rod-shaped BChl c aggregates were sometimes observed. These results strongly suggest that CsmA binds the BChl a in Chlorobium-type chlorosomes and further indicate that none of the nine other chlorosome envelope proteins are absolutely required for maintaining the shape and integrity of chlorosomes. Quantitative estimates suggest that chlorosomes contain approximately equimolar amounts of CsmA and BChl a and that roughly one-third of the surface of the chlorosome is covered by CsmA.

The composition, abundance and apparent molecular masses of chlorosome polypeptides from Chlorobium tepidum and Chlorobium vibrioforme 8327 were compared. The most abundant, low-molecular-mass chlorosome polypeptides of both strains had similar electrophoretic mobilities and abundances, but several of the larger proteins were different in both apparent mass and abundance. Polyclonal antisera raised against recombinant chlorosome proteins of Cb. tepidum recognized the homologous proteins in Cb. vibrioforme, and a one-to-one correspondence between the chlorosome proteins of the two species was confirmed. As previously shown [Ormerod et al. (1990) J Bacteriol 172: 1352–1360], acetylene strongly suppressed the synthesis of bacteriochlorophyll c in Cb. vibrioforme strain 8327. No correlation was found between the bacteriochlorophyll c content of cells and the cellular content of chlorosome proteins. Nine of ten chlorosome proteins were detected in acetylene-treated cultures, and the chlorosome proteins were generally present in similar amounts in control and acetylene-treated cells. These results suggest that the synthesis of chlorosome proteins and the assembly of the chlorosome envelope is constitutive. It remains possible that the synthesis of bacteriochlorophyll c and its insertion into chlorosomes might be regulated by environmental parameters such as light intensity.

Detection of immunoglobulin M (IgM) antibodies has long been used as an important diagnostic tool for identifying active viral infections, but their relevance in later stages has not been clearly definedin vivo. In this study, we followed the kinetics, longevity, and function of influenza virus-specific IgM antibodies for 2 years following sublethal infection of mice with live mouse-adapted A/PR/8/34 virus or immunization with formalin-inactivated virus. These groups mounted robust protective immune responses and survived lethal challenges with 50× 50% lethal dose (LD50) mouse-adapted A/PR/8/34 virus 600 days after the primary exposure. Surprisingly, the virus-specific IgM antibodies persisted along with IgG antibodies, and we found a significantly higher number of IgM-positive (IgM+) virus-specific plasma cells than IgG+plasma cells that persisted for at least 9 months postexposure. The IgM antibodies were functional as they neutralized influenza virus in the presence of complement just as well as IgG antibodies did.

A recombinant Hc fragment of botulinum neurotoxin, serotype B (rBoNTB(Hc)), has been successfully expressed in a Mut+ strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. The fermentation process consisted of batch phase on glycerol, followed by glycerol and methanol fed-batch phases yielding a final cell mass of 60 g/L (dcw) and was easily scaled-up to 60 L. A multistep ion-exchange chromatographic purification process was employed to produce 99% pure Hc fragment. The final yield of the purified antigen was 390 mg per kilogram of wet cell mass. The purified Hc fragment of serotype B was stable, elicited an immune response in mice, and protected upon challenge with native botulin.

Recent studies have demonstrated the effectiveness of vaccine delivery to the skin by vaccine-coated microneedles; however there is little information on the effects of adjuvants using this approach for vaccination. Here we investigate the use of TLR ligands as adjuvants with skin-based delivery of influenza subunit vaccine. BALB/c mice received 1 µg of monovalent H1N1 subunit vaccine alone or with 1 µg of imiquimod or poly(I:C) individually or in combination via coated microneedle patches inserted into the skin. Poly(I:C) adjuvanted subunit influenza vaccine induced similar antigen-specific immune responses compared to vaccine alone when delivered to the skin by microneedles. However, imiquimod-adjuvanted vaccine elicited higher levels of serum IgG2a antibodies and increased hemagglutination inhibition titers compared to vaccine alone, suggesting enhanced induction of functional antibodies. In addition, imiquimod-adjuvanted vaccine induced a robust IFN-γ cellular response. These responses correlated with improved protection compared to influenza subunit vaccine alone, as well as reduced viral replication and production of pro-inflammatory cytokines in the lungs. The finding that microneedle delivery of imiquimod with influenza subunit vaccine induces improved immune responses compared to vaccine alone supports the use of TLR7 ligands as adjuvants for skin-based influenza vaccines.

Previously, a modified HIV Env protein with a heterologous membrane anchor was found to be incorporated into HIV virus-like particles (VLPs) at 10-fold-higher levels than those of unmodified Env. To further improve the immunogenicity of such VLPs, membrane-anchored forms of bacterial flagellin (FliC) or a flagellin with a truncated variable region (tFliC) were constructed to be incorporated into the VLPs as adjuvants. HIV-specific immune responses induced by the resulting VLPs were determined in a guinea pig model. The VLPs induce enhanced systemic antibody responses by either systemic or mucosal vaccination and enhanced mucosal immunity by a mucosal immunization route, as demonstrated by high levels of HIV-specific serum IgG and mucosal IgG and IgA. The quality of the antibody responses was also improved, as shown by enhanced neutralization capacity. VLPs incorporating FliC were more effective in inducing systemic responses, while VLPs containing tFliC were more effective in inducing mucosal IgA responses. The IgG titers in sera were found to last for at least 5 months without a significant drop. These results indicate that HIV VLPs incorporating high levels of Env and a molecular adjuvant have excellent potential for further development as a prophylactic HIV vaccine., IMPORTANCE A prophylactic vaccine is urgently needed to control the spread of HIV/AIDS. Antigens inducing strong systemic and mucosal immune responses are promising as vaccines for this mucosally transmitted disease. We found that novel HIV virus-like particles (VLPs) presenting a high level of Env in its native membrane-bound form and coincorporating an innate immune-signaling adjuvant in the same particles were effective in inducing enhanced systemic and mucosal immunity. As new HIV vaccine candidates, these VLPs bridge the gaps of the innate and adaptive, as well as systemic and mucosal, immune responses, providing a new approach for HIV vaccine development.

Intracellular trafficking of membrane-coated vesicles represents a fundamental process that controls the architecture of different intracellular compartments and communication between the cell and its environment. Major trafficking pathways consist of an inward flux of endocytic vesicles from the plasma membrane and an outward flux of exocytic vesicles to the plasma membrane. This overview describes a number of molecular biology tools commonly used to analyze endocytic and exocytic pathways. The overall emphasis is on major proteins responsible for vesicle formation, recognition, and fusion. These include components of vesicle coats, adaptor complexes, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and Rab guanosine 5'-triphosphatases (GTPases), which represent attractive targets for genetic manipulation aimed at unraveling mechanisms of endocytosis and exocytosis.

Intracellular trafficking of membrane-coated vesicles represents a fundamental process that controls the architecture of different intracellular compartments and communication between the cell and its environment. Major trafficking pathways consist of an inward flux of endocytic vesicles from the plasma membrane and an outward flux of exocytic vesicles to the plasma membrane. This overview describes a number of molecular biology tools commonly used to analyze endocytic and exocytic pathways. The overall emphasis is on major proteins responsible for vesicle formation, recognition, and fusion. These include components of vesicle coats, adaptor complexes, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and Rab guanosine 5'-triphosphatases (GTPases), which represent attractive targets for genetic manipulation aimed at unraveling mechanisms of endocytosis and exocytosis.

The composition, abundance and apparent molecular masses of chlorosome polypeptides from Chlorobium tepidum and Chlorobium vibrioforme 8327 were compared. The most abundant, low-molecular-mass chlorosome polypeptides of both strains had similar electrophoretic mobilities and abundances, but several of the larger proteins were different in both apparent mass and abundance. Polyclonal antisera raised against recombinant chlorosome proteins of Cb. tepidum recognized the homologous proteins in Cb. vibrioforme, and a one-to-one correspondence between the chlorosome proteins of the two species was confirmed. As previously shown [Ormerod et al. (1990) J Bacteriol 172: 1352-1360], acetylene strongly suppressed the synthesis of bacteriochlorophyll c in Cb. vibrioforme strain 8327. No correlation was found between the bacteriochlorophyll c content of cells and the cellular content of chlorosome proteins. Nine of ten chlorosome proteins were detected in acetylene-treated cultures, and the chlorosome proteins were generally present in similar amounts in control and acetylene-treated cells. These results suggest that the synthesis of chlorosome proteins and the assembly of the chlorosome envelope is constitutive. It remains possible that the synthesis of bacteriochlorophyll c and its insertion into chlorosomes might be regulated by environmental parameters such as light intensity.

Intestinal inflammation is associated with epithelial damage and formation of mucosal wounds. Epithelial cells migration is required for wound closure. In inflammatory status, migrating epithelial cells are exposed to proinflammatory cytokines such as IFN-γ. However, influence of such cytokines on intestinal epithelial wound closure remains unknown. The present study was designed to investigate the effect of IFN-γ on migration of model T84 intestinal epithelial cells and recovery of epithelial wounds. IFN-γ significantly inhibited rate of T84 cell migration and closure of epithelial wounds. This effect was accompanied by the formation of large aberrant lamellipodia at the leading edge as well as significant decrease in the number of β1 integrin containing focal adhesions. IFN-γ exposure increased endocytosis of β1 integrin and shifted its accumulation from early/recycling endosomes at the leading edge to a yet unidentified compartment at the cell base. This redirection in β1 integrin transcytosis was inhibited by depolymerization of microtubules with nocodazole and was unaffected by stabilization of microtubules with docetaxel. These results suggest that IFN-γ attenuates epithelial wound closure by microtubule-dependent redirection of β1 integrin transcytosis from the leading edge of migrating cells thereby inhibiting adequate turnover of focal adhesion complexes and cell migration.

Chlorosomes of the green sulfur bacterium Chlorobium tepidum have previously been shown to contain at least 10 polypeptides [Chung, S., Frank, G., Zuber, H., and Bryant, D. A. (1994) Photosynth. Res. 41, 261-275]. Based upon the N-terminal amino acid sequences determined for two of these proteins, the corresponding genes were isolated using degenerate oligonucleotide hybridization probes. The csmI and csmJ genes encode proteins of 244 and 225 amino acids, respectively. A third gene, denoted csmX, that predicts a protein of 221 amino acids with strong sequence similarity to CsmI and CsmJ, was found to be encoded immediately upstream from the csmJ gene. All three proteins have strong sequence similarity in their amino-terminal domains to [2Fe-2S] ferredoxins of the adrenodoxin/putidaredoxin subfamily of ferredoxins. CsmI and CsmJ were overproduced in Escherichia coli, and both proteins were shown by EPR spectroscopy to contain iron-sulfur clusters. The g-tensor and relaxation properties are consistent with their assignment as [2Fe-2S] clusters. Isolated chlorosomes were also shown to contain [2Fe-2S] clusters whose properties were similar to those of the recombinant CsmI and CsmJ proteins. Redox titration of isolated chlorosomes showed these clusters to have potentials of about -201 and +92 mV vs SHE. The former potential is similar to that measured by redox titration of the clusters in inclusion bodies of CsmJ. Possible roles for these iron-sulfur proteins in electron transport and light harvesting are discussed.

Chlorosomes are the light-harvesting antenna complexes of green phototrophic bacteria. Their cores are composed of highly aggregated Bchls (see (1) and (2) for reviews), and the chlorosome envelope is believed to be a monolayer membrane (3) stabilized by proteins (4, 5, 6). Chlorobium tepidum chlorosomes have been shown to contain 10 proteins (4). Genes encoding nine of these proteins have been cloned and characterized (4, 5, 6, 7; D. A. Bryant and V. L. Stirewalt, unpublished results). CsmA (6.0 kDa), CsmB (7.5 I(Da), CsmC (14.3 kDa), CsmD (11.1 kDa), CsmE (6.7 kDa), and CsmH have been shown to be at least partially exposed on the chlorosome envelope (5, 6, unpublished results). The two largest chlorosome proteins, Csml (25.9 kDa) and CsmJ (23.9 kDa), each contain a [2Fe-2S] cluster and are also components of the envelope (E. V. Vassilieva, B. L. Zybailov, M. L. Antonkine, F. Yang, C. Jakobs, J. H. Golbeck, and D. A. Bryant, manuscript in preparation). In this study, the treatment of Cb. tepidum chlorosomes with the detergents SDS, Lubrol PX, and Triton X-100 resulted in selective protein extraction and Bchl c de-oligomerization. The data provide further insight into the structural organization of the proteins of chlorosomes.