Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria’s proteome was achieved using Beckman Coulter’s Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Epilithic and Endolithic Bacterial Communities in Limestone from a Maya Archaeological Site

Biodeterioration of archaeological sites and historic buildings is a major concern for conservators, archaeologists, and scientists involved in preservation of the world's cultural heritage. The Maya archaeological sites in southern Mexico, some of the most important cultural artifacts in the Western Hemisphere, are constructed of limestone. High temperature and humidity have resulted in substantial microbial growth on stone surfaces at many of the sites. Despite the porous natureof limestone and the common occurrence of endolithic microorganisms in many habitats, little is known about the microbial flora living inside the stone. We found a large endolithic bacterial community in limestone from the interior of the Maya archaeological site Ek' Balam. Analysis of 16S rDNA clones demonstrated disparate communities (endolithic: >80% Actinobacteria, Acidobacteria, and Low GC Firmicutes; epilithic: >50% Proteobacteria). The presence of differing epilithic and endolithic bacterial communities may be a significant factor for conservation of stone cultural heritage materials and quantitative prediction of carbonate weathering.     

Effects of AeDNV infection on Aedes aegypti lifespan and reproduction

The effects of Aedes Densovirus (AeDNV) infections on survival, fertility, fecundity and vertical transmission in Aedes aegypti (Diptera: Culicidae) were measured in laboratories in Kiev, Ukraine and Colorado, USA and incorporated into a predictive model of the effects of AeDNV on vector capacity. Adult lifespan and daily survival were reduced in AeDNV infected mosquitoes. This effect was dependent on the dose of the virus. Infected females had decreased fecundity. The oviposition rate was less in infected females and the hatch rate declined in eggs laid by infected females. The amounts of AeDNV in infected females and the infection rate of their offspring were measured with real-time PCR. The average filial transmission rate was 70% and larval infection rates from infected females varied between 42 and 62%. Vertically infected larvae, and individual eggs contained 1 × 10⁵ AeDNV genome equivalents (geq). Modeling the effects of AeDNV infection on Ae. aegypti populations suggested a large decrease in the numbers of eggs, larvae, pupae, and adults arising from infected mothers and suggested that AeDNV treatment of larvae could cause up to a 76% reduction of infectious mosquito days.     

Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background

We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner.

Methodology/Principal Findings

Optical imaging of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes.

Conclusions/Significance

These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors.     

Molecular identification of lyso-glycerophosphocholines as endogenous immunosuppressives in bovine and rat gonadal fluids

The ability of the gametes to escape detection by the immune system is vital to successful human reproduction. Furthermore, the observed capacity of the testis in some species to support tissue grafts without rejection (immunological privilege) indicates that spermatogenic cells are protected by local immunoregulatory mechanisms. One of these mechanisms involves targeting T cells for inactivation and destruction within the testicular environment. Although the fluids of the testis and ovary surrounding the developing gametes contain soluble factors that inhibit T cells, the identity of the molecule(s) responsible for this activity has been unknown. Using a specific T-cell proliferation assay to monitor bioactivity, these molecules were purified from bovine ovarian follicular fluid by methanol extraction and sequential reverse-phase HPLC (RP-HPLC). All purified active fractions coincided with the elution position on RP-HPLC of several small molecules ranging in size from 496 to 522 Da. The same molecules were localized to the immunosuppressive fractions of rat testicular interstitial fluid. The active molecules were identified, using capillary electrophoresis electrospray ionization mass spectroscopy, as lyso-glycerophosphocholines (lyso-GPCs), namely, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, a 18:2a/lyso-GPC (putatively, 1-linoleoyl-sn-glycero-3-phosphocholine), and a 20:4a/lyso-GPC (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine). Comparison of the bioactivity and mass spectroscopy profiles of two of the purified molecules with their synthetic standards confirmed the identification. These molecules inhibit T-cell proliferation in response to activation and induce apoptosis of these cells in a time- and dose-dependent manner. The emergence of gonadal lyso-GPCs as potential regulators of critical immune events opens up new avenues of inquiry into the origins of autoimmune infertility and more generally into mechanisms of peripheral immunoregulation and the development of novel immunosuppressives.     

Shaking a leg and hot to trot: the effects of body size and temperature on running speed in ants

Abstract.  1. Data were compiled from the literature and our own studies on 24 ant species to characterise the effects of body size and temperature on forager running speed.
2. Running speed increases with temperature in a manner consistent with the effects of temperature on metabolic rate and the kinetic properties of muscles.
3. The exponent of the body mass-running speed allometry ranged from 0.14 to 0.34 with a central tendency of approximately 0.25. This body mass scaling is consistent with both the model of elastic similarity, and a model combining dynamic similarity with available metabolic power.
4. Even after controlling for body size or temperature, a substantial amount of inter-specific variation in running speed remains. Species with certain lifestyles [e.g. nomadic group predators, species which forage at extreme (>60 °C) temperatures] may have been selected for faster running speeds.
5. Although ants have a similar scaling exponent to mammals for the running speed allometry, they run slower than predicted compared with a hypothetical mammal of similar size. This may in part reflect physiological differences between invertebrates and vertebrates.