Deinococcus radiodurans UWO298 Dependence on Background Radiation for Optimal Growth.

Ionizing radiation is a major environmental variable for cells on Earth, and so organisms have adapted to either prevent or to repair damages caused by it, primarily from the appearance and accumulation of reactive oxygen species (ROS). In this study, we measured the differential gene expression in Deinococcus radiodurans UWO298 cultures deprived of background ionizing radiation (IR) while growing 605 m underground at the Waste Isolation Pilot Plant (WIPP), reducing the dose rate from 72.1 to 0.9 nGy h-1 from control to treatment, respectively. This reduction in IR dose rate delayed the entry into the exponential phase of the IR-shielded cultures, resulting in a lower biomass accumulation for the duration of the experiment. The RNASeq-based transcriptome analysis showed the differential expression of 0.2 and 2.7% of the D. radiodurans genome after 24 and 34 h of growth in liquid culture, respectively. Gene expression regulation after 34 h was characterized by the downregulation of genes involved in folding newly synthesized and denatured/misfolded proteins, in the assimilation of nitrogen for amino acid synthesis and in the control of copper transport and homeostasis to prevent oxidative stress. We also observed the upregulation of genes coding for proteins with transport and cell wall assembly roles. These results show that D. radiodurans is sensitive to the absence of background levels of ionizing radiation and suggest that its transcriptional response is insufficient to maintain optimal growth.

The Phenotypic and Transcriptomic Response of the Caenorhabditis elegans Nematode to Background and Below-Background Radiation Levels.

Studies of the biological effects of low-level and below-background radiation are important in understanding the potential effects of radiation exposure in humans. To study this issue we exposed the nematode Caenorhabditis elegans to average background and below-background radiation levels. Two experiments were carried-out in the underground radiation biology laboratory at the Waste Isolation Pilot Plant (WIPP) in New Mexico USA. The first experiment used naïve nematodes with data collected within 1 week of being placed underground. The second experiment used worms that were incubated for 8 months underground at below background radiation levels. Nematode eggs were placed in two incubators, one at low radiation (ca.15.6 nGy/hr) and one supplemented with 2 kg of natural KCl (ca. 67.4 nGy/hr). Phenotypic variables measured were: (1) egg hatching success (2) body size from larval development to adulthood, (3) developmental time from egg to egg laying adult, and (4) egg laying rate of young adult worms. Transcriptome analysis was performed on the first experiment on 72 h old adult worms. Within 72 h of being underground, there was a trend of increased egg-laying rate in the below-background radiation treatment. This trend became statistically significant in the group of worms exposed to below-background radiation for 8 months. Worms raised for 8 months in these shielded conditions also had significantly faster growth rates during larval development. Transcriptome analyses of 72-h old naïve nematode RNA showed significant differential expression of genes coding for sperm-related proteins and collagen production. In the below-background radiation group, the genes for major sperm protein (msp, 42% of total genes) and sperm-related proteins (7.5%) represented 49.5% of the total genes significantly up-regulated, while the majority of down-regulated genes were collagen (col, 37%) or cuticle-related (28%) genes. RT-qPCR analysis of target genes confirmed transcriptomic data. These results demonstrate that exposure to below-background radiation rapidly induces phenotypic and transcriptomic changes in C. elegans within 72 h of being brought underground.

Removal of antibiotic resistance genes in an algal-based wastewater treatment system employing Galdieria sulphuraria: A comparative study.

In this study, we compared removal of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) in two wastewater treatment systems fed with the same primary effluent: a conventional wastewater treatment system (consisting of a trickling filter followed by an activated sludge process) versus an algal-based system, employing an extremophilic alga, Galdieria sulphuraria. Our results demonstrated that the algal system can reduce concentrations of erythromycin- and sulfamethoxazole-resistant bacteria in the effluent more effectively than the conventional treatment system. A decreasing trend of total bacteria and ARGs was observed in both the treatment systems. However, the relative ratio of most ARGs (qnrA, qnrB, qnrS, sul1) and intI1 in the surviving bacteria increased in the conventional system; whereas, the algal system reduced more of the relative abundance of qnrA, qnrS, tetW and intⅠ1 in the surviving bacteria. The role of bacteriophages in horizontal gene transfer (HGT) of ARGs in the two systems was indicated by a positive correlation between ARG absolute abundance in bacteriophage and ARG relative abundance in the bacteria. Four of the five detectable genes (qnrS, tetW, sul1 and intI1) were significantly reduced in the algal system in bacteriophage phase which signified a decrease in phage-mediated ARG transfer in the algal system. Results of this study demonstrate the feasibility of the algal-based wastewater treatment system in decreasing ARGs and ARB and in minimizing the spread of antibiotic resistance to the environment.

Effects of turbulent aggregation on clay floc breakup and implications for the oceanic environment.

Understanding how turbulence impacts marine floc formation and breakup is key to predicting particulate carbon transport in the ocean. While floc formation and sinking rate has been studied in the laboratory and in-situ, the breakup response to turbulence has attracted less attention. To address this problem, the breakup response of bentonite clay particles flocculated in salt water was studied experimentally. Flocs were grown in a large aggregation tank under unmixed and mixed aggregation conditions and then subjected to turbulent pipe flow. Particle size was quantified using microscope imaging and in-situ measurements obtained from standard optical oceanographic instruments; a Sequoia Scientific LISST-100X and two WET Labs ac-9 spectrophotometers. The LISST instrument was found to capture the breakup response of flocs to turbulent energy, though the resulting particle size spectra appear to have underestimated the largest floc lengthscales in the flow while overestimating the abundance of primary particles. Floc breakup and the resulting shift towards smaller particles caused an increase in spectral slope of attenuation as measured by the ac-9 instruments. The Kolmogorov lengthscale was not found to have a limiting effect on floc size in these experiments. While the flocs were found to decrease in overall strength over the course of the two-month experimental time period, repeatable breakup responses to turbulence exposure were observed. Hydrodynamic conditions during floc formation were found to have a large influence on floc strength and breakup response. A non-constant strength exponent was observed for flocs formed with more energetic mixing. Increased turbulence from mixing during aggregation was found to increase floc fractal dimension and apparent density, resulting in a shift in the breakup relationships to higher turbulence dissipation rates. The results suggest that marine particle aggregation and vertical carbon transport concepts should include the turbulence energy responsible for aggregate formation and the resulting impact on floc strength, density, and the disruption potential.

Transcriptome analysis reveals a stress response of Shewanella oneidensis deprived of background levels of ionizing radiation.

Natural ionizing background radiation has exerted a constant pressure on organisms since the first forms of life appeared on Earth, so that cells have developed molecular mechanisms to avoid or repair damages caused directly by radiation or indirectly by radiation-induced reactive oxygen species (ROS). In the present study, we investigated the transcriptional effect of depriving Shewanella oneidensis cultures of background levels of radiation by growing the cells in a mine 655 m underground, thus reducing the dose rate from 72.1 to 0.9 nGy h-1 from control to treatment, respectively. RNASeq transcriptome analysis showed the differential expression of 4.6 and 7.6% of the S. oneidensis genome during early- and late-exponential phases of growth, respectively. The greatest change observed in the treatment was the downregulation of ribosomal proteins (21% of all annotated ribosomal protein genes during early- and 14% during late-exponential) and tRNA genes (14% of all annotated tRNA genes in early-exponential), indicating a marked decrease in protein translation. Other significant changes were the upregulation of membrane transporters, implying an increase in the traffic of substrates across the cell membrane, as well as the up and downregulation of genes related to respiration, which could be interpreted as a response to insufficient oxidants in the cells. In other reports, there is evidence in multiple species that some ROS not just lead to oxidative stress, but act as signaling molecules to control cellular metabolism at the transcriptional level. Consistent with these reports, several genes involved in the metabolism of carbon and biosynthesis of amino acids were also regulated, lending support to the idea of a wide metabolic response. Our results indicate that S. oneidensis is sensitive to the withdrawal of background levels of ionizing radiation and suggest that a transcriptional response is required to maintain homeostasis and retain normal growth.

Extending the applicability of the four-flux radiative transfer method.

A generalized four-flux method capable of modeling and tuning the spectral reflectance of a diverse range of complex composite coatings is presented. An example application is exploring and maximizing the visible and near-infrared (IR) spectral reflectance available from the diverse structures arising from combinations of the many practical paint ingredients that are available or can be made when applied to different substrates. This requires consideration of scatterers that can differ in composition, particle size, size distribution, and fill factor, and are held in place by a variety of organic binders, which typically partially absorb in the near IR. This extended model is further enhanced by an explicit matrix algorithm that allows analysis of diverse multilayer stacks. This is applied to a multilayer and is designed to model useful changes that result from varying the pigment fill factor as a function of depth within a layer. What we believe is a novel feature is the way the scattering affects matrix absorptance. The model includes contributions to total absorptance from the scattering pigments and from the paint binder that can arise in different bands or simultaneously at the same wavelengths. Model accuracy is demonstrated by example results when compared to experimental data on dried single layer paint profiles using imaged cross sections. The model input covering the actual pigment and binder properties used are material, shape, size, and size distributions, mass added, and the measured optical constants from 400 nm to 2,500 nm of the undoped binder resin layer. One interesting result is the comparison of a two-layered stack, with bigger particles in the first layer and smaller ones in the second, to one with the opposite depth profile.

Enzymatic Pre-treatment of Wastewater to Minimize Recovery by Reverse Transcriptase PCR of RNA from Inactive Bacteriophages.

Quantitative viral risk assessments for wastewaters are notoriously difficult. The often considered quantitative reverse transcriptase PCR reflects poorly on virus infectivity rates leading to inaccurate risk interpretations. Various techniques focused on the degradation of the nucleic acids of non-infective viruses were previously employed. We comparatively assessed the effectiveness of such enzymatic treatments for MS2 bacteriophage in treated wastewaters. The single use of RNase A at an appropriate concentration may be as effective as the combination of RNase followed by Proteinase K and more rapid. While all tested enzymatic treatments minimized recovery of RNA (>95 %) in the absence of infective MS2, none completely eliminated the signal recovery. Selection of any enzymatic protocol for minimizing recovery of RNA from degraded, non-infective viruses should balance the methods efficacy with its expediency.

Short RNA indicator sequences are not completely degraded by autoclaving.

Short indicator RNA sequences (<100 bp) persist after autoclaving and are recovered intact by molecular amplification. Primers targeting longer sequences are most likely to produce false positives due to amplification errors easily verified by melting curves analyses. If short indicator RNA sequences are used for virus identification and quantification then post autoclave RNA degradation methodology should be employed, which may include further autoclaving.

Application of carbon nanotube technology for removal of contaminants in drinking water: a review.

Carbon nanotube (CNT) adsorption technology has the potential to support point of use (POU) based treatment approach for removal of bacterial pathogens, natural organic matter (NOM), and cyanobacterial toxins from water systems. Unlike many microporous adsorbents, CNTs possess fibrous shape with high aspect ratio, large accessible external surface area, and well developed mesopores, all contribute to the superior removal capacities of these macromolecular biomolecules and microorganisms. This article provides a comprehensive review on application of CNTs as adsorbent media to concentrate and remove pathogens, NOM, and cyanobacterial (microcystin derivatives) toxins from water systems. The paper also surveys on consideration of CNT based adsorption filters for removal of these contaminants from cost, operational and safety standpoint. Based on the studied literature it appears that POU based CNT technology looks promising, that can possibly avoid difficulties of treating biological contaminants in conventional water treatment plants, and thereby remove the burden of maintaining the biostability of treated water in the distribution systems.

The preparation of a plasmonically resonant VO2 thermochromic pigment.

Vanadium dioxide (VO(2)) undergoes a reversible metal-insulator transition, normally at approximately 68 degrees C. While the properties of continuous semi-transparent coatings of VO(2) are well known, there is far less information available concerning the potential use of discrete VO(2) nanoparticles as a thermochromic pigment in opaque coatings. Individual VO(2) nanoparticles undergo a localized plasmon resonance with near-infrared light at about 1100 nm and this resonance can be switched on and off by simply varying the temperature of the system. Therefore, incorporation of VO(2) nanoparticles into a coating system imbues the coating with the ability to self-adaptively modulate its own absorptive efficiency in the near-infrared. Here we examine the magnitude and control of this phenomenon. Prototype coatings are described, made using VO(2) powder produced by an improved process. The materials are characterized using calorimetry, x-ray diffraction, high-resolution scanning electron microscopy, transmission electron microscopy, and by measurement of optical properties.

Adsorption of Bacillus subtilis on single-walled carbon nanotube aggregates, activated carbon and NanoCeram.

Adsorption equilibrium and kinetics of Bacillus subtilis spores on single-walled carbon nanotube aggregates were investigated to explore the possibility of using single-walled carbon nanotubes for concentration, detection and removal of pathogens from contaminated water sources. Batch adsorption experiments were conducted to determine adsorption kinetics and adsorption equilibrium of B. subtilis spores on single-walled carbon nanotube aggregates, activated carbon and NanoCeram. The adsorption kinetics data were analyzed with both the Lagergren pseudo first order and a pseudo second order models. The adsorption equilibrium data on three porous media were quantified by the Henry's law constant. It was observed that both the Lagergren first order rate model and the pseudo second order model correlate the adsorption kinetic data well although the calculated adsorption rate constants vary with adsorbate concentrations. The Henry's law adsorption equilibrium constant of B. subtilis spores on single-walled carbon nanotube aggregates is about 27-37 times higher than those on activated carbon and NanoCeram. The high adsorption affinity of carbon nanotubes towards the B. subtilis spores is due to the mesoporous structure and unique surface properties of carbon nanotubes. These results suggest that single-walled carbon nanotube aggregates are good candidates as biosensors and adsorbent media for concentrating, detecting and removal of pathogens from contaminated water resources.

Adsorption kinetics of Escherichia coli and Staphylococcus aureus on single-walled carbon nanotube aggregates.

Batch adsorption studies to determine adsorption kinetics of Escherichia coli (E.coli) K12 and Staphylococcus aureus (S.aureus) SH 1000 bacterial cells on single-walled carbon nanotube aggregates were performed at two different initial concentrations. The diffusivity of E. coli cells in single-walled carbon nanotube aggregates obtained was 6.54 x 10(-9) and 8.98 x 10(-9) cm(2)/s, whereas that of S. aureus was between 1.00 x 10(-7) and 1.66 x 10(-7) cm(2)/s respectively. In addition to batch adsorption studies, electron microscopy studies were also conducted. The results suggest that diffusion kinetics of bacterial cells is concentration dependent as well as bacteria dependent. Diffusivity of S. aureus is two orders of magnitude greater than E. coli cells. This proves to be beneficial from an adsorption perspective where it is desired to filter microorganisms (water pretreatment and wastewater post treatment) and from nanotube biosensor perspective where it is desired to simultaneously capture and detect biothreat agents in a shorter span of time.

The development of point-of-use water filters as sampling devices in bioforensics: extent of microbial sorption and elution.

The foundational idea for this project is that household faucet-mounted water filters may be used as bioforensic sampling devices to detect the extent of a potential bioagent release in domestic water supplies. An optimized eluent solution was determined experimentally by quantifying recoveries of microorganisms from point-of-use (POU) drinking water filters. The optimized extraction protocol was then used in mock bioagent release experiments to determine the feasibility of POU filters as bioforensic sampling devices. Bacillus atrophaeus spores, Escherichia coli and PP7 virus were exposed to filters and the number of attached organisms was determined by enumerating the unattached organisms on selective agar media. Subsequently, the filters were eluted and the percent of extracted organisms was determined based on the number of attached organisms. Two popular brands of carbon block filters retained 92%-99% of representative virus, spore and vegetative bacteria. In back-flush elutions of single filters, the most efficient eluent was identified as a combination of 1% peptone and 1% Tween-80, and extraction recovered 25.4% (+/-17.5%) of attached E. coli, 20.4% (+/-3.6%) of B. atrophaeus spores, and 9.4% (+/-5.2%) of PP7 virions (+/- standard deviations). In bioagent release studies in which filters were challenged with 100 agents mL(-1), greater than 99% of the spores were retained by the filters, and the percent of attached spores that were recovered ranged from 10.4% at day 0 to 4.3% five days after the release event (averaged from five separate experiments). In contrast, E. coli, Salmonella typhimurium and PP7 virus were rapidly inactivated in the chlorinated tap water, indicating their improbable survival in chlorinated water supplies. It is therefore concluded that household water filters can be used as microbial sampling devices for bioforensic applications in the event of a bioagent release in domestic drinking water supplies.

Constituents with independence from growth temperature for bacteria using pyrolysis-gas chromatography/differential mobility spectrometry with analysis of variance and principal component analysis.

Four bacteria, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus warneri, and Micrococcus luteus, were grown at temperatures of 23, 30, and 37 degrees C and were characterized by pyrolysis-gas chromatography/differential mobility spectrometry (Py-GC/DMS) providing, with replicates, 120 data sets of retention time, compensation voltage, and ion intensity, each for negative and positive polarity. Principal component analysis (PCA) for 96 of these data sets exhibited clusters by temperature of culture growth and not by genus. Analysis of variance was used to isolate the constituents with dependences on growth temperature. When these were subtracted from the data sets, Fisher ratios with PCA resulted in four clusters according to genus at all temperatures for ions in each polarity. Comparable results were obtained from unsupervised PCA with 24 of the original data sets. The ions with taxonomic features were reconstructed into 3D plots of retention time, compensation voltage, and Fisher ratio and were matched, through GC-mass spectrometry (MS), with chemical standards attributed to the thermal decomposition of proteins and lipid A. Results for negative ions provided simpler data sets than from positive ions, as anticipated from selectivity of gas phase ion-molecule reactions in air at ambient pressure.

Analysis of bacteria by pyrolysis gas chromatography-differential mobility spectrometry and isolation of chemical components with a dependence on growth temperature.

Pyrolysis gas chromatography-differential mobility spectrometry (py-GC-DMS) analysis of E. coli, P. aeruginosa, S. warneri and M. luteus, grown at temperatures of 23, 30, and 37 degrees C, provided data sets of ion intensity, retention time, and compensation voltage for principal component analysis. Misaligned chromatographic axes were treated using piecewise alignment, the impact on the degree of class separation (DCS) of clusters was minor. The DCS, however, was improved between 21 to 527% by analysis of variance with Fisher ratios to remove chemical components independent of growth temperature. The temperature dependent components comprised 84% of all peaks in the py-GC-DMS analysis of E. coli and were attributed to the pyrolytic decomposition of proteins rather than lipids, as anticipated. Components were also isolated in other bacteria at differing amounts: 41% for M. luteus, 14% for P. aeruginosa, and 4% for S. warneri, and differing patterns suggested characteristic dependence on temperature of growth for these bacteria. These components are anticipated to have masses from 100 to 200 Da by inference from differential mobility spectra.

Analysis of bacterial strains with pyrolysis-gas chromatography/differential mobility spectrometry.

Eight vegetative bacterial strains and two spores were characterized by pyrolysis-gas chromatography with differential mobility spectrometry (py-GC/DMS) yielding topographic plots of ion intensity, retention time, and compensation voltage simultaneously for ions in positive and negative polarity. Biomarkers were found in the pyrolysate at characteristic retention times and compensation voltages and were confirmed by standard addition with GC/MS analyses providing discrimination between Gram negative and Gram positive bacterial types, but no recognition of individual strains within the Gram negative bacteria. Principal component analysis was applied using two dimensional data sets of ion intensity versus retention time at five compensation voltages including the reactant ion peaks all in positive and negative ion polarity. Clustering was observed with compensation voltage (CV) chromatograms associated with ion separation in the DMS detector and little or no clustering was observed with the reactant ion peaks or CV chromatograms where ion separation is poor. Consistent clustering of Gram positive B. odysseyi and Gram negative E. coli in both positive and negative polarities with the reactant ion peak chromatograms and key CV chromatograms suggests common but unknown common chemical compositions in the pyrolysate.

Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation.

The UL32 gene of human cytomegalovirus (CMV) encodes a prominent betaherpesvirus-conserved virion tegument protein, called pp150 (basic phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion adjacent to the nucleus at late times during infection. Using a UL32 deletion mutant (DeltaUL32-BAC) (where BAC is bacterial artificial chromosome), we demonstrate that pp150 is critical for virion maturation in the cytoplasmic compartment. Cotransfection of a pp150 expression plasmid with DeltaUL32-BAC DNA led to complementation of the replication defect with focus formation due to secondary spread. Deletion of the amino terminus of pp150 or disruption of the betaherpesvirus conserved regions, CR1 and CR2, revealed these regions to be critical for replication. In contrast, deletion of the carboxyl terminus only partially compromised maturation while disruption of glycosylation sites had no effect. An African green monkey CMV UL32 homolog complemented DeltaUL32-BAC replication but murine CMV M32 failed to complement, consistent with evolutionary divergence of rodent and primate cytomegaloviruses. Infection with DeltaUL32-BAC showed normal expression of all kinetic classes of viral genes and replication of viral DNA, with accumulation of viral DNA-containing particles in the cytoplasm; however, mutant virus did not spread to adjacent cells. In contrast to this block in virion infectivity, cell-to-cell transfer of pp65-containing particles was observed, suggesting that release of dense bodies continued in the absence of pp150. These observations demonstrate that pp150 is critical for virion egress, possibly at the stage of final envelopment.

Contribution of GADD45 family members to cell death suppression by cellular Bcl-xL and cytomegalovirus vMIA.

Mammalian cells and viruses encode inhibitors of programmed cell death that localize to mitochondria and suppress apoptosis initiated by a wide variety of inducers. Mutagenesis was used to probe the role of a predicted alpha-helical region within the hydrophobic antiapoptotic domain (AAD) of cytomegalovirus vMIA, the UL37x1 gene product. This region was found to be essential for cell death suppression activity. A screen for proteins that interacted with the AAD of functional vMIA but that failed to interact with mutants identified growth arrest and DNA damage 45 (GADD45alpha), a cell cycle regulatory protein activated by genotoxic stress, as a candidate cellular binding partner. GADD45alpha interaction required the AAD alpha-helical character that also dictated GADD45alpha-mediated enhancement of death suppression. vMIA mutants that failed to interact with GADD45alpha were completely nonfunctional in cell death suppression, and any of the three GADD45 family members (GADD45alpha, GADD45beta/MyD118, or GADD45gamma/OIG37/CR6/GRP17) was able to cooperate with vMIA; however, none influenced cell death when introduced into cells alone. GADD45alpha was found to increase vMIA protein levels comparably to treatment with protease inhibitors MG132 and ALLN. Targeted short interfering RNA knockdown of all three GADD45 family members maximally reduced vMIA activity, and this reduction was abrogated by additional GADD45alpha. Interestingly, GADD45 family members were also able to bind and enhance cell death suppression by Bcl-xL, a member of the Bcl-2 family of cell death suppressors, suggesting a direct cooperative link between apoptosis and the proteins that regulate the DNA damage response.

Mitochondrial cell death suppressors carried by human and murine cytomegalovirus confer resistance to proteasome inhibitor-induced apoptosis.

Human cytomegalovirus carries a mitochondria-localized inhibitor of apoptosis (vMIA) that is conserved in primate cytomegaloviruses. We find that inactivating mutations within UL37x1, which encodes vMIA, do not substantially affect replication in TownevarATCC (Towne-BAC), a virus that carries a functional copy of the betaherpesvirus-conserved viral inhibitor of caspase 8 activation, the UL36 gene product. In Towne-BAC infection, vMIA reduces susceptibility of infected cells to intrinsic death induced by proteasome inhibition. vMIA is sufficient to confer resistance to proteasome inhibition when expressed independent of viral infection. Murine cytomegalovirus m38.5, whose position in the viral genome is analogous to UL37x1, exhibits mitochondrial association and functions in much the same manner as vMIA in inhibiting intrinsic cell death. This work suggests a common role for vMIA in rodent and primate cytomegaloviruses, modulating the threshold of virus-infected cells to intrinsic cell death.

Interaction between constitutively expressed heat shock protein, Hsc 70, and cysteine string protein is important for cortical granule exocytosis in Xenopus oocytes.

In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by approximately 80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a protein that is important for regulated exocytosis. Because the NH2-terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes.