Movement of a tritium plume in shallow groundwater at a legacy low-level radioactive waste disposal site in eastern Australia.

Between 1960 and 1968 low-level radioactive waste was buried in a series of shallow trenches near the Lucas Heights facility, south of Sydney, Australia. Groundwater monitoring carried out since the mid 1970s indicates that with the exception of tritium, no radioactivity above typical background levels has been detected outside the immediate vicinity of the trenches. The maximum tritium level detected in ground water was 390 kBq/L and the median value was 5400 Bq/L, decay corrected to the time of disposal. Since 1968, a plume of tritiated water has migrated from the disposal trenches and extends at least 100 m from the source area. Tritium in rainfall is negligible, however leachate from an adjacent and fill represents a significant additional tritium source. Study data indicate variation in concentration levels and plume distribution in response to wet and dry climatic periods and have been used to determine pathways for tritium migration through the subsurface.

Characterization of foliar manganese (Mn) in Mn (hyper)accumulators using X-ray absorption spectroscopy.

Plant hyperaccumulation of the essential nutrient manganese (Mn) is a rare phenomenon most evident in the Western Pacific region, and differs from hyperaccumulation of other elements. Mn hyperaccumulators employ a variety of species-dependent spatial distribution patterns in sequestering excess foliar Mn, including primary sequestration in both nonphotosynthetic and photosynthetic tissues. This investigation employed synchrotron X-ray absorption spectroscopy (XAS) in a comparative study of Mn (hyper)accumulators, to elucidate in situ the chemical form(s) of foliar Mn in seven woody species from Australia, New Caledonia and Japan. Foliar Mn was found to predominate as Mn(II) in all samples, with strong evidence of the role of carboxylic acids, such as malate or citrate, as complexing ligands. Overall, the X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine-structure spectroscopy (EXAFS) data appeared weighted against previous observations that oxalate binds excess Mn in Mn-(hyper)accumulating species.

Manganese accumulation in the leaf mesophyll of four tree species: a PIXE/EDAX localization study.

Little is known about the spatial distribution of excess manganese (Mn) in the leaves of tolerant plants. Recently, the first such study of a Mn hyperaccumulator showed that the highest localized Mn concentrations occur in the photosynthetic tissue. This is in contrast to reports based on localization of foliar accumulation of other heavy metals. Here, four tree species, Gossia bidwillii, Virotia neurophylla, Macadamia integrifolia and Macadamia tetraphylla, which hyperaccumulate or strongly accumulate Mn, were studied. Cross-sectional foliar Mn localization was carried out in situ using proton-induced X-ray emission/energy dispersive X-ray analysis (PIXE/EDAX). All four species contained photosynthetic tissues with multiple palisade layers. These were shown to be the primary sequestration sites for Mn. Mn was not detected in the epidermal tissues. The findings of this study demonstrate a concurrence of three traits in four tree species, that is, accumulation of excess Mn in the leaves, its primary sequestration in the photosynthetic tissues, and multiple-layer palisade mesophyll.

Insights into biological functions across species: examining the role of Rab proteins in YIP1 family function.

The YIP1 family comprises an evolutionarily conserved group of membrane proteins, which share the ability to bind di-prenylated Rab proteins. The biochemical capability of YIP1 family proteins suggests a possible role in the cycle of physical localization of Rab proteins between their cognate membranes and the cytosol. YIP1 is essential for viability in yeast and a deletion of YIP1 can be rescued with the human homologue YIP1A. We have made use of this evolutionary conservation of function to generate a series of mutant alleles of YIP1 to investigate the biological role of Yip1p. Our findings indicate evidence for the participation of Yip1p in both Rab and COPII protein function; at present, we are not able to distinguish between the models that these roles represent, i.e. independent or dependent activities of Yip1p.

Determination of metal--EDTA complexes in soil solution and plant xylem by ion chromatography-electrospray mass spectrometry.

An ion chromatography-electrospray mass spectrometry (IC-MS) method was developed to quantify the metal complexes of ethylenediaminetetraacetic acid (EDTA) in soil solution and plant xylem exudate. Suitable separation of the metal-EDTA complexes was achieved on a Dionex AS5 column using 2 mM Na2CO3 as the eluant. However, satisfactory detection by eluant suppressed IC-MS, in either the positive or negative ion detection mode, could not be attained. A new eluant that still attained suitable separation and produced ionic species that could be detected by MS in the negative ion mode was developed. The eluant consisted of 2.5 mM (NH4)2CO3, 9.7 mM NH4OH, and 4% (v/v) methanol and had a pH 9.9. Even though eluant suppressed IC-MS degraded detection limits by a factor of 4 over the nonsuppressed system, using the retention time and not the m/z (mass-to-charge ratio) of the intact chelate for identification, the latter allowed the metal complexes to be detected intact and was optimized for the analysis of environmental samples. The number of metal-EDTA species that could be detected was limited by the eluant used for ion chromatography (i.e. only those complexes that were stable at high pH), with metal-EDTA complexes of Al, Cd, Cu, Co, Mn, Ni, Pb, and Zn being adequately resolved. Iron(III), Ca, MgEDTA, and EDTA itself were not detected. Detection limits for the various complexes ranged from 0.1 to 1 microM.

Yop1p, the yeast homolog of the polyposis locus protein 1, interacts with Yip1p and negatively regulates cell growth.

Rab proteins are small GTPases that are essential elements of the protein transport machinery of eukaryotic cells. Each round of membrane transport requires a cycle of Rab protein nucleotide binding and hydrolysis. We have recently characterized a protein, Yip1p, which appears to play a role in Rab-mediated membrane transport in Saccharomyces cerevisiae. In this study, we report the identification of a Yip1p-associated protein, Yop1p. Yop1p is a membrane protein with a hydrophilic region at its N terminus through which it interacts specifically with the cytosolic domain of Yip1p. Yop1p could also be coprecipitated with Rab proteins from total cellular lysates. The TB2 gene is the human homolog of Yop1p (Kinzler, K. W., Nilbert, M. C., Su, L.-K., Vogelstein, B., Bryan, T. M., Levey, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M. S., Altschul, S. F., Horii, A., Ando, H. M., Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991) Science 253, 661-665). Our data demonstrate that Yop1p negatively regulates cell growth. Disruption of YOP1 has no apparent effect on cell viability, while overexpression results in cell death, accumulation of internal cell membranes, and a block in membrane traffic. These results suggest that Yop1p acts in conjunction with Yip1p to mediate a common step in membrane traffic.

Identification of a Sec4p GTPase-activating protein (GAP) as a novel member of a Rab GAP family.

A yeast open reading frame sharing homology with the two known yeast Rab GTPase-activating proteins (GAPs), Gyp6p and Gyp7p, was found in a data base search. We have named the gene containing this open reading frame GYP1. Recombinant Gyp1p showed GAP activity on Sec4p, increasing both its steady-state rate and single turnover GTPase activity. Gyp1p also stimulated the GTPase activity of several other yeast Rab proteins including Ypt1p, Ypt7p, and Ypt51p but showed no GAP activity on Ypt6p and Ypt32p. Deletion of the GYP1 gene or overexpression of Gyp1p did not alter the growth rate of yeast. However, overexpression of Gyp1p was inhibitory in combination with a subset of secretory mutants including sec4-8 and several ypt1 mutants. This effect is probably due to the increase in GAP activity, which can be observed in a lysate from cells overexpressing Gyp1p. The finding that yeast Rab GAPs share homology with proteins in other species, such as Caenorhabditis elegans and human, suggests the existence of a conserved Rab GAP family.

Interactions of nucleotide release factor Dss4p with Sec4p in the post-Golgi secretory pathway of yeast.

SEC4 is an essential gene encoding a small GTPase that is involved in Golgi to cell surface transport in Saccharomyces cerevisiae and is a paradigm for studies on the mode of action of Rab proteins. We describe here the features of interaction of Sec4p with the accessory protein Dss4p. Dss4p is found both on membranes and in the cytosol; however, it is the membrane fraction that is complexed to Sec4p. Dss4p, like its mammalian counterpart, Mss4, binds zinc, and disruption of the zinc-binding site disrupts the ability of the protein to interact with Sec4p. DSS4 overexpression can rescue the lethal phenotype of two alleles of SEC4, corresponding to dominant mutations of Ras. We demonstrate that this suppression is due to the ability of Dss4p to form a tight complex with the mutant forms of Sec4p and hence sequester the mutant protein from its inhibitory effect. These results imply an in vivo role for Dss4p as a guanine nucleotide dissociation stimulator. In vitro the protein has the ability to stimulate the dissociation rate of both GDP and GTP from Sec4p. We examined the relationship of GDI1 and DSS4 with SEC4 both genetically and biochemically. These results exclude a role for DSS4 in the recruitment of Sec4p/GDI onto membranes.

Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles.

The small GTPase Sec4p is required for vesicular transport at the post-Golgi stage of yeast secretion. Here we present evidence that mutations in SEC2, itself an essential gene that acts at the same stage of the secretory pathway, cause Sec4p to mislocalize as a result of a random rather than a polarized accumulation of vesicles. Sec2p and Sec4p interact directly, with the nucleotide-free conformation of Sec4p being the preferred state for interaction with Sec2p. Sec2p functions as an exchange protein, catalyzing the dissociation of GDP from Sec4 and promoting the binding of GTP. We propose that Sec2p functions to couple the activation of Sec4p to the polarized delivery of vesicles to the site of exocytosis.

Non-clinical roles of Army family physicians in their first post-residency assignment and their level of preparedness for these roles.

A cross-sectional survey of the 1992 and 1993 graduates from Army Family Practice Residencies was done to determine their non-clinical roles and their perceived level of preparedness for these roles. Sixty-nine of the 98 questionnaires mailed (70%) were returned. The non-clinical roles were divided into command, administrative, committee (subdivided into hospital level and department/clinic level), and duties outside of the hospital (subdivided into military and administrative). Twenty-nine (42%) had commands, 58 (84%) had administrative roles, 48 (70%) had hospital committee roles, 53 (77%) had department/clinic committee roles, 54 (78%) had military roles, and 42 (61%) had administrative roles outside of the hospital or clinic. The self-reported level of preparedness, on a five point Likert scale (1 = not at all, 3 = adequately, 5 = superbly), ranged from 2.2 for command to 2.9 for administrative roles outside the hospital. Residents did not feel adequately prepared for their non-clinical roles.

Sphingolipid transport in mitotic HeLa cells.

Mitotic and interphase HeLa cells were labeled with [3H]serine. Ceramide and its derivatives, lactosylceramide and sphingomyelin, were biosynthetically labeled under both conditions. Only in the absence of nocodazole, as the cells entered telophase, was an additional glycosphingolipid synthesized which was identified as GA2 (GalNAc(beta 1,4)Gal(beta 1,4)Glc(beta 1,1)Cer). Ceramide, the basic sphingolipid precursor, is synthesized in the endoplasmic reticulum, whereas its immediate derivatives are synthesized in early Golgi compartments. Transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi is inhibited in mitotic cells while ceramide acquires early Golgi modifications under the same conditions, suggesting that ceramide can be delivered to the Golgi by a different route. Since GA2 is synthesized in late Golgi, its absence in mitotic cells strongly argues for an in vivo inhibition of intra-Golgi transport, an observation with important implications for the mechanism of Golgi division.