Paleozoic cave system preserves oldest-known evidence of amniote skin.

The richest and most diverse assemblage of early terrestrial tetrapods is preserved within the infilled cave system of Richards Spur, Oklahoma (289-286 Mya1). Some of the oldest-known terrestrial amniotes2,3 are exquisitely preserved here because of early impregnation and encasement of organic material by oil-seep hydrocarbons within rapidly deposited clay-rich cave sediments under toxic anoxic conditions.4 This phenomenon has also afforded the preservation of exceedingly rare integumentary soft tissues, reported here, providing critical first evidence into the anatomical changes marking the transition from the aquatic and semiaquatic lifestyles of anamniotes to the fully terrestrial lifestyles of early amniotes. This is the first record of a skin-cast fossil (3D carbonization of the skin proper) from the Paleozoic Era and the earliest known occurrence of epidermal integumentary structures. We also report on several compression fossils (carbonized skin impressions), all demonstrating similar external morphologies to extant crocodiles. A variety of previously unknown ossifications, as well as what are likely palpebral ossifications of the deeper dermis layer of the skin, are also documented. These fossils also serve as invaluable references for paleontological reconstructions. Chromatographic analysis of extractable hydrocarbons from bone and cave samples indicates that the source rock is the Devonian age Woodford Shale. Hydrocarbons derived from ancient marine organisms interacting with geologically younger terrestrial vertebrates have therefore resulted in the oldest-known preservation of amniote skin proper.

Evaluating Local and Directional Resolution of Cryo-EM Density Maps.

A systematic and quantitative evaluation of cryo-EM maps is necessary to judge their quality and to capture all possible sources of error. A single value for global resolution is insufficient to accurately describe the quality of a reconstructed density. We describe the estimation and evaluation of two additional resolution measures, local and directional resolution, using methods based on the Fourier shell correlation (FSC). We apply the protocol to samples that encompass different types of pathologies a user is expected to encounter and provide analyses on how to interpret the output files and resulting maps. Implementation of these tools will facilitate density interpretation and can guide the user in adapting their experiments to improve the quality of cryo-EM maps, and by extension atomic models.

Geochemical characterization and renal cell toxicity of water-soluble extracts from U.S. Gulf Coast lignite.

An assortment of organic material can leach from lignite (low-rank coal) in water, and the water-soluble fraction from lignite has been associated with adverse health effects in areas of the Balkans. Recent efforts have been made to evaluate this hypothesis in other areas where lignite is in contact with groundwater like in the U.S. Gulf Coast region. In this study, five Gulf Coast lignite samples were extracted with water, and the water-soluble portion of the coal was then characterized by total organic carbon, UV-Vis spectroscopy, and gas chromatography/mass spectrometry. Additionally, human kidney cells (HK-2) were exposed to water-soluble extracts of Gulf Coast lignite to assess toxicity. Cell viability was measured, and a dose-response curve was used to generate IC50 values that ranged from 490 to 3000 ppm. The most toxic extract (Dolet Hills) was from Louisiana where lignite-derived organic material has been previously linked to high incidence of renal pelvic cancer. Concentrations of nephrotoxic metals (As, Cd, Co, Cu, Hg, Pb, V, Zn) were screened and were below those considered toxic to renal cells. We conclude that leachates from lignite do indeed have toxic affects on cultured human renal cells. Although the IC50 values are higher than the concentration of organic matter in the local groundwater, typically < 5 ppm, the effects of long-term low-level exposure is not known.

ESRD and ESRD-DM associated with lignite-containing aquifers in the U.S. Gulf Coast region of Arkansas, Louisiana, and Texas.

Balkan endemic nephropathy (BEN) is an irreversible, lethal kidney disease that occurs in regions of the Balkans where residents drink untreated well water. A key factor contributing to the development of BEN may be consumption of dissolved organic matter leached from low-rank coal called lignite. This hypothesis-known as lignite-water hypothesis-was first posed for areas of the Balkans. It is possible that a BEN-like condition exists in the United States (US) Gulf Coast region in parts of the Mississippi Embayment and the Texas Coastal Uplands aquifers -Arkansas, Louisiana, and Texas, for instance-that rely heavily on groundwater from aquifers that contain lignite. This study utilizes a geographic information system (GIS) to map the distributions of end-stage renal disease (ESRD) in relation to water from lignite-containing aquifers in the tri-state region. Regional patterns emerged from geospatial analysis, suggesting that counties that relied on lignite-containing aquifers for their main water source had higher rates of ESRD in comparison to other populations in the region that rely on other water sources, including surface water and groundwater from aquifers not associated with lignite seams. Statewide rates of ESRD and diabetes associated ESRD (ESRD-DM) showed strong correlations to the percent of families at or below poverty level and the percentage of African Americans. These confounding factors somewhat mitigate the association seen between ESRD and lignite-containing regions at the state level. However, at the larger tri-state view, there is a significant (p = 0.002) increase in incidence rates where groundwater is connected to lignite-containing aquifers when considering both race and poverty. Additionally, no relationship was observed between the rate of public water supply withdrawal from lignite-bearing aquifers and rates of ESRD or ESRD-DM at the state or tri-state regions, supporting the observation that the risk associated with water from lignite-containing aquifers is limited to water from untreated domestic supply.

Carbon Isotope Fractionation of 1,2-Dibromoethane by Biological and Abiotic Processes.

1,2-Dibromethane (EDB) is a toxic fuel additive that likely occurs at many sites where leaded fuels have impacted groundwater. This study quantified carbon (C) isotope fractionation of EDB associated with anaerobic and aerobic biodegradation, abiotic degradation by iron sulfides, and abiotic hydrolysis. These processes likely contribute to EDB degradation in source zones (biodegradation) and in more dilute plumes (hydrolysis). Mixed anaerobic cultures containing dehalogenating organisms (e.g., Dehaloccoides spp.) were examined, as were aerobic cultures that degrade EDB cometabolically. Bulk C isotope enrichment factors (εbulk) associated with biological degradation covered a large range, with mixed anaerobic cultures fractionating more (εbulk from -8 to -20‰) than aerobic cultures (εbulk from -3 to -6‰). εbulk magnitudes associated with the abiotic processes (dihaloelimination by FeS/FeS2 and hydrolysis) were large but fairly well constrained (εbulk from -19 to -29‰). As expected, oxidative mechanisms fractionated EDB less than dihaloelimination and substitution mechanisms, and biological systems exhibited a larger range of fractionation, potentially due to isotope masking effects. In addition to quantifying and discussing εbulk values, which are highly relevant for quantifying in situ EDB degradation, an innovative approach for constraining the age of EDB in the aqueous phase, based on fractionation during hydrolysis, is described.

Thiosteranes in samples impacted by fecal materials and their potential use as marker of sewage input.

Sewage impacted soil, sludge and water samples were studied to understand the occurrence and formation of thiosteranes and to determine the relevance of these compounds as tracers for sewage input into the environment. Soils were collected from wastewater irrigation fields (Wroclaw, Poland), water from the Nexapa River Basin (Mexico), which also received wastewater and wastewater treatment plant(WWTP) effluent, and water and sludge from the Norman WWTP (USA) at different treatment stages. Thiosteranes represented a high proportion of the steroid fraction in the Wroclaw irrigation field and the Nexapa River Basin samples. Small amounts of thiosteranes were found in anaerobically digested sludge from the Norman WWTP. A good correlation between coprostanone and thiosterane concentrations suggests thiosteranes were produced by stanone sulphurization under anoxic conditions. Thiosterane stability under anoxic and suboxic conditions indicates their potential use as tracers for environmental input of sewage products or land application of sewage sludge.

The use of sterol distributions combined with compound specific isotope analyses as a tool to identify the origin of fecal contamination in rivers.

The sterol distributions of 9 sediment samples from the Illinois River Basin (OK and AR, USA) were examined in order to identify the source of fecal contamination. The samples were extracted with organic solvent using sonication and the fractions containing the sterols were isolated and analyzed by gas chromatography-mass spectrometry. The sterol distributions of the Illinois River samples were dominated by phytosterols. They were compared to those of different animal feces and manures using a principal component analysis and correspondence appeared between the sediments and one group of chicken feces samples. Gas chromatography-isotope ratio mass spectrometry analyses were also performed to determine the δ(13)C values for the phytosterols and to get an indication of their origin based on the C(3)/C(4) plant signatures. The δ(13)C values obtained ranged from -30.6 ‰ to -17.4 ‰ (VPDB) corresponding to a mixed signature between C(3) and C(4) plants, indicating a C(4) plant contribution to the C(3) plant natural background. These observations indicate that a proportion of the phytosterols originated from chicken feces.

Validation of adsorbents for sample preconcentration in compound-specific isotope analysis of common vapor intrusion pollutants.

Isotope ratios of volatile organic compounds (VOCs) in the environment are often of interest in contaminant fate studies. Adsorbent preconcentration-thermal desorption of VOCs can be used to collect environmental vapor samples for compound-specific isotope analysis (CSIA). While active adsorbent samplers offer logistic benefits in handling large volumes of air, their performance in preserving VOCs isotope ratios was not previously tested under sampling conditions corresponding to typical indoor air sampling conditions. In this study, the performance of selected adsorbents was tested for preconcentration of TCE (for determination of C and Cl isotope ratios), PCE (C and Cl) and benzene (C and H). The key objective of the study was to identify the adsorbent(s) permitting preconcentration of the target VOCs present in air at low µg/m(3) concentrations, without significant alteration of their isotope ratios. Carboxen 1016 was found to perform well for the full range of tested parameters. Carboxen 1016 can be recommended for sampling of TCE, PCE and benzene, for CSIA, from air volumes up to 100 L. Variable extent of isotope ratio alteration was observed in the preconcentration of the target VOCs on Carbopack B and Carbopack X, resulting from partial analyte loss via adsorbent bed breakthrough and (possibly) via incomplete desorption. The results from testing the Carbopack B and Carbopack X highlight the need of adsorbent performance validation at conditions fully representative of actual sample collection conditions, and caution against extrapolation of performance data toward more challenging sampling conditions.

Impacts of microbial community composition on isotope fractionation during reductive dechlorination of tetrachloroethylene.

Isotope fractionation has been used with increasing frequency as a tool to quantify degradation of chlorinated aliphatic pollutants in the environment. The objective of this research was to determine if the electron donor present in enrichment cultures prepared from uncontaminated sediments influenced the extent of isotope fractionation of tetrachloroethylene (PCE), either directly, or through its influence on microbial community composition. Two PCE-degrading enrichment cultures were prepared from Duck Pond (DP) sediment and were incubated with formate (DPF) or H(2) (DPH) as electron donor. DPF and DPH were significantly different in both product distribution and extent of isotope fractionation. Chemical and isotope analyses indicated that electron donors did not directly affect the product distribution or the extent of isotope fractionation for PCE reductive dechlorination. Instead, restriction fragment length polymorphism (RFLP) and sequence analysis of the 16S rRNA clone libraries of DPF and DPH identified distinct microbial communities in each enrichment culture, suggesting that differences in microbial communities were responsible for distinct product distributions and isotope fractionation between the two cultures. A dominant species identified only in DPH was closely related to known dehalogenating species (Sulfurospirillum multivorans and Sulfurospirillum halorespirans) and may be responsible for PCE degradation in DPH. Our study suggests that different dechlorinators exist at the same site and can be preferentially stimulated by different electron donors, especially over the long-term (i.e., years), typical of in-situ ground water remediation.

Biodegradation of low-molecular-weight alkanes under mesophilic, sulfate-reducing conditions: metabolic intermediates and community patterns.

We evaluated the ability of the native microbiota in a low-temperature, sulfidic natural hydrocarbon seep (Zodletone) to metabolize short-chain hydrocarbons. n-Propane and n-pentane were metabolized under sulfate-reducing conditions in initial enrichments and in sediment-free subcultures. Carbon isotope analysis of residual propane in active enrichments showed that propane became enriched in (13)C by 6.7 (+/-2.0) per thousand, indicating a biological mechanism for propane loss. The detection of n-propylsuccinic and isopropylsuccinic acids in active propane-degrading enrichments provided evidence for anaerobic biodegradation via a fumarate addition pathway. A eubacterial 16S rRNA gene survey of sediment-free enrichments showed that the majority of the sequenced clones were phylogenetically affiliated within the Deltaproteobacteria. Such sequences were most closely affiliated with clones retrieved from hydrocarbon-impacted marine ecosystems, volatile fatty acid metabolizers, hydrogen users, and with a novel Deltaproteobacterial lineage. Other cloned sequences were affiliated with the Firmicutes and Chloroflexi phyla. The sequenced clones were only distantly (<95%) related to other reported low-molecular-weight alkane-degrading sulfate-reducing populations. This work documents the potential for anaerobic short-chain n-alkane metabolism for the first time in a terrestrial environment, provides evidence for a fumarate addition mechanism for n-propane activation under these conditions, and reveals microbial community members present in such enrichments.

The relative contributions of abiotic and microbial processes to the transformation of tetrachloroethylene and trichloroethylene in anaerobic microcosms.

Reductive dechlorination of tetrachloroethylene (PCE) and trichloroethylene (TCE) was studied in well-defined microcosms prepared with aquifer materials from three locations. Electron donors and terminal electron acceptors were added to both stimulate microbial activity and generate reactive minerals via microbial iron and sulfate reduction. The relative importance of abiotic and microbial PCE and TCE reductive dechlorination was then assessed by analysis of reaction products and kinetics and, in some cases, by stable carbon isotope fractionation. The predominant PCE and TCE transformation pathway in most microcosms was microbial reductive dechlorination. Rates of abiotic transformation were similar in magnitude to those for microbial reductive dechlorination in only a few cases where the activity of dechlorinating bacteria was low. Comparison of geochemical conditions with abiotic product recoveries showed thatthe greatest extent of abiotic reductive dechlorination occurred under iron- and sulfate-reducing conditions. Under these two geochemical conditions, high concentrations of Fe(II) and S(-II) solid species were present, suggesting the involvement of Fe(II) and S(-II) minerals in abiotic reductive dechlorination. Both abiotic and microbial dechlorination of PCE and TCE took place under almost all microcosm conditions; the relative rates of the two processes under field conditions will depend on factors such as the abundance of dechlorinating bacteria, soil properties, and the mass loading of reactive minerals.

Kinetic and isotope analyses of tetrachloroethylene and trichloroethylene degradation by model Fe(II)-bearing minerals.

The kinetics and in some cases stable carbon isotope fractionation associated with abiotic reductive dechlorination of tetrachloroethylene (PCE) and trichloroethylene (TCE) by model Fe(II)-bearing minerals present in anaerobic soils were measured. The minerals studied were chloride green rust (GR-Cl), sulfate green rust (GR-SO(4)), pyrite, magnetite, and adsorbed Fe(II) or FeS formed at the surface of goethite by treatment with dissolved Fe(II) or S(-II). We observed some abiotic transformation of PCE and TCE in every system studied, as evidenced by the presence of abiotic reaction products. Bulk enrichment factors (epsilon(bulk) values) for TCE transformation by GR-Cl and pyrite were -23.0+/-1.8 per thousand and -21.7+/-1.0 per thousand, respectively, which are more negative than reported values for microbial TCE dechlorination and could provide one means for distinguishing microbial from abiotic dechlorination of TCE in the environment. Considering the time scale of subsurface remediation technologies, including natural attenuation, minerals such as green rusts, pyrite, and magnetite have the potential to contribute to the transformation of PCE and TCE at contaminated sites.

Distinguishing abiotic and biotic transformation of tetrachloroethylene and trichloroethylene by stable carbon isotope fractionation.

Significant carbon isotope fractionation was observed during FeS-mediated reductive dechlorination of tetrachloroethylene (PCE) and trichloroethylene (TCE). Bulk enrichment factors (E(bulk)) for PCE were -30.2 +/- 4.3 per thousand (pH 7), -29.54 +/- 0.83 per thousand (pH 8), and -24.6 +/- 1.1 per thousand (pH 9). For TCE, E(bulk) values were -33.4 +/- 1.5 per thousand (pH 8) and -27.9 +/- 1.3 per thousand (pH 9). A smaller magnitude of carbon isotope fractionation resulted from microbial reductive dechlorination by two isolated pure cultures (Desulfuromonas michiganensis strain BB1 (BB1) and Sulfurospirillum multivorans (Sm)) and a bacterial consortium (BioDechlor INOCULUM (BDI)). The E(bulk) values for biological PCE microbial dechlorination were -1.39 +/- 0.21 per thousand (BB1), -1.33 +/- 0.13 per thousand (Sm), and -7.12 +/- 0.72 per thousand (BDI), while those for TCE were -4.07 +/- 0.48 per thousand (BB1), -12.8 +/- 1.6 per thousand (Sm), and -15.27 +/- 0.79 per thousand (BDI). Reactions were investigated by calculation of the apparent kinetic isotope effect for carbon (AKIEc), and the results suggest that differences in isotope fractionation for abiotic and microbial dechlorination resulted from the differences in rate-limiting steps during the dechlorination reaction. Measurement of more negative E(bulk) values at sites contaminated with PCE and TCE may suggest the occurrence of abiotic reductive dechlorination by FeS.

A comparison of the metal content of some benthic species from coastal waters of the Florida panhandle using high-resolution inductively coupled plasma mass spectrometry (ICP-MS) analysis.

Benthic marine invertebrates, sediment, and water from several locations along the Florida panhandle coast from St. Joseph Bay in the west to the mouth of the Wakulla River in the east, including from several river estuaries, were analyzed by double focusing ICP-MS (Finnigan MAT ELEMENT) for Cd, Hg, Pb, Cu, Zn, and As. All were detected in all samples. Sponges generally contained higher levels of Cd than other species. Microciona prolifera sponges from St. Joseph Bay had higher As levels (8.1-13.6 microg/g dry weight) than sponges collected from Dickerson Bay or Appalachee Bay (2.20-9.7) and higher Cd levels (0.43-0.73) than that of a single Microciona specimen collected from Dickerson Bay (0.29). Water content of As was about 20-30x higher in St. Joseph Bay than in any other location, and sediment levels of Cd were about 9x higher. Cu and Zn were higher in organic sediment from St. Joseph Bay than they were in other areas. The Pb content of several sponge species and two of tunicates was considerably higher than in other species. The uptake of most metals in this study (except As) appeared to be affected by the metal, genus, species, and location as much as by levels in either water or sediment. In general, sponges and tunicates seemed to accumulate higher levels than most other species, possibly a function of high filtration rates. The ICP-MS method is useful for environmental studies, but the instrument requires considerable maintenance.

Effects of experimental manipulation of pH and salinity on Cd(2+) uptake by the sponge Microciona prolifera and on sponge cell aggregation induced by Ca(2+) and Cd(2+).

Marine sponges (Microciona prolifera) collected in St. Joseph Bay, Florida panhandle, were exposed for 2 h to pH/salinity unit combinations of 7.4/30, 6.3/30, 7.4/11, and 6.3/11. Cell suspensions from these were aggregated with 24 microM of either CaCl(2) or CdCl(2). Cells exposed to the low/low (11/6.3) combination aggregated spontaneously (no added stimulus) in 8/11 experiments, suggesting a significant disturbance of normal function, possibly involving disrupted ion uptake. In all other combinations aggregation proceeded normally and there were no statistically significant differences among the groups. CdCl(2) was as effective an aggregation stimulus as CaCl(2). The calcium channel blocker verapamil (100 microM) reduced calcium-induced aggregation by 15% but had no effect on cadmium (Cd)-induced aggregation, indicating that L-type calcium channels do not play a major role in aggregation induced by these divalent cations. Sponge tissue was exposed for 48 h to the same pH/salinity combinations but containing Cd (15 or 150 microg/ml) and then dried and analyzed for Cd. All sponges concentrated Cd but those exposed to low salinity concentrated it most (in one case x13). Low pH alone had no appreciable effect but appeared to increase the effect of low salinity. One sponge with a native Cd content of 47.2 microg/g dry weight had the highest acquired Cd content. The results of this study indicate that low levels of salinity and pH, similar to those we recorded in the study area, facilitate the accumulation of Cd, but not via L-type calcium channels, and disrupt normal aggregation responses of the cell. These results may help explain a previous observation that cells from M. prolifera from this area, with high native levels of Cd, failed to aggregate in response to CaCl(2)[Philp RB (1999) Comp Biochem Physiol 124C:41-49] and also the frequent die-offs of Microciona that have virtually eliminated this sponge from its local habitat.

Cadmium content of the marine sponge Microciona prolifera, other sponges, water and sediment from the eastern Florida panhandle: possible effects on Microciona cell aggregation and potential roles of low pH and low salinity.

Marine sponges from shallow bays and estuaries along the Florida panhandle contained cadmium (Cd), 1.78-27.9 microg g(-1) (dry weight). Levels > 7 microg g(-1) were never found in a variety of other benthic invertebrates. Water levels of Cd were of the order 0.22-0.43 microg ml(-1) and did not differ significantly between the two study areas (Panacea and St. Joseph Bay). Cd in organic sediment however, was significantly (P = 0.033) higher around Panacea (0.343 +/- 0.063 S.E.M.) than in St. Joseph Bay (0.18 +/-0.223 S.E.M.). Salinity of 10 and pH 6.5 were recorded following heavy rains, and can constitute a hostile environment for these organisms. Temporal variations of Cd and Cu but not Zn were observed in Microciona prolifera over a period of 2 years in St. Joseph Bay, and a negative correlation was observed between Cd content and the Ca(2+)-induced aggregation of Microciona cells suspended in Ca2+/Mg(2+)-free artificial seawater. Cells unresponsive to Ca2+ aggregated in response to releasers of Ca2+ from internal stores (thapsigargin, A23187 and tetredecylamine), suggesting that they were viable but dormant. In vitro studies showed that Cd2+ is about equipotent with Ca2+ in inducing aggregation of Microciona cells, whereas lower concentrations (1-2 x 10(-3) M), added 10 min before inducing aggregation with CaCl2, inhibited aggregation, possibly by blocking Ca2+ uptake. Microciona contained a metallothionein (MT)-like protein. Taken together these results suggest that naturally-acquired Cd may interfere with cell regulatory processes in Microciona. Possible effects of low pH and salinity on Cd uptake and detrimental effects on cell function are discussed.

Nitrous oxide enhances Na+/Ca++ exchange in the neuroblastoma cell line SK-N-SH.

Changes in the concentration of cytosolic free calcium ([Ca++]i) play fundamental roles in the initiation and regulation of many neuronal processes. Altered regulation of [Ca++]i has been implicated in the action of some anesthetics. We investigated the effects of nitrous oxide (N2O) on Ca++ mobilization and membrane potential in the human neuroblastoma cell line SK-N-SH. [Ca++]i was monitored by fluorescence spectrophotometry of cells loaded with fura-2 or fluo-3. N2O reversibly suppressed carbachol-stimulated increases in [Ca++]i. N2O also inhibited increases in [Ca++]i induced by calcium ionophore or depolarization suggesting a mechanism involving enhanced efflux or sequestration of cytosolic Ca++. The inhibitory effect of N2O was attenuated when the transmembrane Na+ gradient was altered either by suspending cells in nominally Na(+)-free buffer or by pretreating cells with ouabain. The inhibitory effect of N2O was also attenuated by the Na+/Ca++ exchange inhibitor 3,4-dichlorobenzamil. The effects of N2O on membrane potential were measured fluorimetrically using bis(1,3-dibutylthiobarbituric acid)-trimethine oxonol. In the presence of N2O, resting membrane potential was hyperpolarized, a condition that would favor Ca++ efflux mediated by the electrogenic Na+/Ca++ exchanger. Taken together, these findings indicate that N2O suppresses carbachol-stimulated increases in [Ca++]i by enhancing Na+/Ca++ exchange activity. Enhancement of neuronal Na+/Ca++ exchange may contribute to the anesthetic action of N2O.

Casein kinase II associates with Egr-1 and acts as a negative modulator of its DNA binding and transcription activities in NIH 3T3 cells.

Although the activation domains within early growth response gene protein 1 (Egr-1) have been mapped, little is known of the kinases which phosphorylate Egr-1 and how phosphorylation correlates with the transcriptional activity of Egr-1. In this study we report that casein kinase II (CKII) co-immunoprecipitates with Egr-1 from NIH 3T3 cell lysates. The association of Egr-1 and CKII requires the C terminus of Egr-1 and CKII phosphorylates Egr-1 in vitro. The in vitro phosphorylation of Egr-1 by CKII and that induced by serum in vivo was compared by examining the CNBr-digested fragments of the phosphorylated Egr-1. CKII strongly phosphorylates fragments 7 and 10 which cover part of the activation/nuclear localization and DNA binding domains of Egr-1. CKII also phosphorylates, albeit weakly, fragments 5 and 8 which cover part of activation domain and the entire repression domain of Egr-1, respectively. Strong phosphorylation on fragment 10 as well as fragment 5 was also observed in Egr-1 immunoprecipitated from serum-induced, 32P-labeled cells. CKII phosphorylation of Egr-1 resulted in a decrease of its DNA binding as well as its transcriptional activities.

GS28, a 28-kilodalton Golgi SNARE that participates in ER-Golgi transport.

Little is known about the integral membrane proteins that participate in the early secretory pathway of mammalian cells. The complementary DNA encoding a 28-kilodalton protein (p28) of the cis-Golgi was cloned and sequenced. The protein was predicted to contain a central coiled-coil domain with a carboxyl-terminal membrane anchor. An in vitro assay for endoplasmic reticulum-Golgi transport was used to show that p28 participates in the docking and fusion stage of this transport event. Biochemical studies established that p28 is a core component of the Golgi SNAP receptor (SNARE) complex.