Fate of indicator microorganisms, Giardia and Cryptosporidium in subsurface flow constructed wetlands.

Limited information is available on the ability of subsurface flow wetlands to remove enteric pathogens. Two multi-species wetlands, one receiving secondary sewage effluent and the other potable (disinfected) groundwater were studied from February 1995 to August 1996, at the Pima County Constructed Ecosystems Research Facility in Tucson, Arizona. Each wetland had a retention time of approximately 4 days. The objectives of this study were (1) to evaluate the ability of multi-species subsurface wetlands to physically remove Giardia cysts; Cryptosporidium oocysts, total and fecal coliforms, and coliphages; and (2) to determine the likely impact of local wildlife on the occurrence of these indicators and pathogens. In the wetland receiving secondary sewage effluent, total coliforms were reduced by an average of 98.8% and fecal coliforms by 98.2%. Coliphage were reduced by an average of 95.2%. Both Giardia cysts and Cryptosporidium oocysts were reduced by an average of 87.8 and 64.2%, respectively. In the wetland receiving disinfected groundwater, an average of 1.3 x 10(2) total coliforms/100 mL and 22.3 fecal coliforms/100 mL were most likely contributed by both flora and fauna. No parasites or coliphages were detected.

Nutrient and heavy metal uptake and storage in constructed wetland systems in Arizona.

The Constructed Ecosystems Research Facility (CERF) was conceived in the early 1980s as a test facility to explore the potential for using plants to treat wastewater in the arid west of the USA. One of the major issues that has been identified in the use of constructed wetland technology is plant nutrient uptake and tissue storage of nutrients as well as heavy metals. Our approach to understanding plant uptake and storage has been to look at both controlled conditions in constructed systems and background concentrations in natural systems. Plant tissues have been collected and analyzed from natural systems and from controlled systems receiving either wastewater or municipal water. Plants studied included the herbaceous species Anemopsis californica (Yerba mansa), Scirpus spp. (bulrush) and Typha domingensis (cattail), and tree species Fraxinus velutina (ash), Populus fremontii (cottonwood) and Salix spp. (willow). Data indicate that uptake varies not only among plant species, but also among chemical species, depending upon water quality within the wetlands. Leaf tissues of Fraxinus, Salix and Populus, contained the lowest amounts of nutrients and heavy metals studied (Na, P, K, Cu, Pb and Zn), while the root tissues of the herbaceous plants generally had the highest concentrations.

In vivo evidence of a role for hepatic lipase in human apoB-containing lipoprotein metabolism, independent of its lipolytic activity.

Hepatic lipase (HL) is a key player in lipoprotein metabolism by modulating, through its lipolytic activity, the triglyceride (TG) and phospholipid content of apolipoprotein B (apoB)-containing lipoproteins and of high density lipoproteins (HDL), thereby affecting their size and density. A new and separate role has been suggested for HL in cellular lipoprotein metabolism, in which it serves as a ligand promoting cellular uptake of apoB-containing remnant lipoproteins and HDL. We tested the hypothesis that HL has both a lipolytic and a nonlipolytic role in human lipoprotein metabolism, by measuring lipid plasma concentrations, lipoprotein density distribution by density gradient ultracentrifugation, and lipoprotein composition, in three subjects with HL deficiency: two of the patients (S-1 and S-3) were characterized as having neither plasma HL activity nor detectable HL protein; the third subject (S-2) had no plasma HL activity but a detectable amount (35.5 ng/ml) of HL protein. All HL-deficient subjects showed a severalfold increase in lipoprotein TG content across the lipoprotein density spectrum [very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and HDL] as compared with control subjects. They also had remarkably more buoyant LDL particles (LDL-R(f) = 0.342;-0.394) as compared with the control subjects (LDL-R(f) = 0.303). Subjects S-1 and S-3 (no HL activity or protein) presented with a distinct increase in cholesterol and apoB levels in the IDL and VLDL density range as compared with patient S-2, with detectable HL protein, and the control subjects. This study provides evidence in humans that HL indeed plays an important role in lipoprotein metabolism independent of its enzymatic activity: in particular, inactive HL protein appears to affect VLDL and IDL particle concentration, whereas HL enzymatic activity seems to influence VLDL-, IDL-, LDL-, and HDL-TG content and their physical properties.

Intussusception in association with childhood cocaine intoxication: a case report.

A case report of an 11-month-old infant with cocaine intoxication and intussusception is presented. We present this case because there are no prior reports in the literature of cocaine intoxication associated with intussusception. The diagnosis was not initially considered in this patient. We feel the association is biologically plausible and alert physicians to consider intussusception in the differential diagnosis of the cocaine-exposed child with vomiting and abdominal pain.

Human cathepsin S: chromosomal localization, gene structure, and tissue distribution.

The human lysosomal cysteine proteinases, cathepsins H, L, and B, have been mapped to chromosomes 15, 9, and 8, respectively, and the genomic structures of cathepsins L and B have been determined. We report here the chromosomal localization and partial gene structure for a recently sequenced human cysteine proteinase, cathepsin S. A 20-kilobase pair genomic clone of the human cathepsin S gene was isolated from a human fibroblast genomic library and used to map the human cathepsin S gene to chromosome 1q21 by fluorescence in situ hybridization. This clone contains exons 1 through 5, introns 1 through 4, part of intron 5, and > 7 kilobase pairs of the 5'-flanking sequence. The gene structure of human cathepsin S is similar to that of cathepsin L through the first 5 exons, except that cathepsin S introns are substantially larger. Sequencing of the 5'-flanking region revealed, similar to human cathepsin B, no classical TATA or CAAT box. In contrast to cathepsin B, cathepsin S contains only two SP1 and at least 18 AP1 binding sites that potentially could be involved in regulation of the gene. This 5'-flanking region also contains CA microsatellites. The presence of AP1 sites and CA microsatellites suggest that cathepsin S can be specifically regulated. Results of Northern blotting using probes for human cathepsins B, L, and S are consistent with this hypothesis; only cathepsin S shows a restricted tissue distribution, with highest levels in spleen, heart, and lung. In addition, immunostaining of lung tissue demonstrated detectable cathepsin S only in lung macrophages. The high level of expression in the spleen and in phagocytes suggests that cathepsin S may have a specific function in immunity, perhaps related to antigen processing.

Incorporation of tubulin from an evolutionarily diverse source, Physarum polycephalum, into the microtubules of a mammalian cell.

Physarum myxamoebal tubulin was injected into PtK2 cells to determine whether tubulin from this eukaryotic microbe could act as a reporter for microtubule growth and dynamics in a mammalian cell. The distribution of Physarum tubulin was determined by the use of a monoclonal antibody specific for Physarum tubulin and unable to detect mammalian tubulin. Physarum tubulin was incorporated into the microtubules of both interphase arrays and the mitotic spindle. Measurements of microtubule turnover kinetics were found to be similar to those of other studies in which chemically modified brain tubulin has been used. Results using this heterologous system demonstrate that tubulin from an evolutionarily diverse organism can be used as a marker for microtubule growth in mammalian cells. Furthermore, the Physarum tubulin was able to endow the injected cells with novel properties. Resistance to colchicine-induced microtubule disassembly, a characteristic of Physarum tubulin, was conferred on the injected PtK2 cells. Use of this heterologous reporter tubulin system has also revealed features of variation in microtubule dynamics both within individual cells and between cells.

A mutant beta-tubulin confers resistance to the action of benzimidazole-carbamate microtubule inhibitors both in vivo and in vitro.

The mutant BEN210 of Physarum polycephalum is highly resistant to a number of benzimidazole carbamate agents, including methylbenzimidazole-2-yl-carbamate and parbendazole. The resistance is conferred by the benD210 mutation in a structural gene for beta-tubulin. This mutant allele encodes a beta-tubulin with novel electrophoretic mobility. We have used this strain to determine whether the mutant beta-tubulin is used in microtubules and whether this usage permits microtubule polymerisation in the presence of drugs both in vivo and in vitro. In vitro assembly studies of tubulin purified from the mutant strain have shown that microtubules are formed both in the absence of drugs and in all drug concentrations tested (up to 50 microM parbendazole). In contrast, the assembly of microtubules from wild-type tubulin in vitro is totally inhibited by 2-5 microM parbendazole. Thus the resistance of BEN210 to parbendazole observed in vivo has been reproduced in vitro using tubulin purified from the mutant strain. Electrophoretic analysis of the microtubules formed in vitro has shown that both the wild-type and the mutant beta-tubulin are incorporated into the microtubules and that the proportion of mutant to wild-type beta-tubulin appears to remain constant with increasing drug concentration. This is the first demonstration of a single mutation in a tubulin structural gene causing an altered function of the gene product in vitro.

Patterns of tubulin isotype synthesis and usage during mitotic spindle morphogenesis in Physarum.

Tubulin synthesis in the naturally synchronous plasmodium of Physarum polycephalum is a markedly periodic event restricted to the late G2 period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle. We have shown that all four tubulin isotypes identified in the plasmodium (alpha 1, alpha 2, beta 1 and beta 2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: beta 2 greater than alpha 1 greater than alpha 2 greater than beta 1. We have also shown that tubulins synthesized in the G2 period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin-containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucleokinesis.

Microtubule formation and the initial association of tubulin dimers.

Microtubule protein could be prepared in high yield, and could form copious microtubules, in solutions containing glutamate but not in solutions containing only phosphate ions. Correspondingly, tubulin after isolation showed an association equilibrium in the presence of glutamate (or other zwitterions), but not in phosphate buffers. The correlation suggests that this association to tetramers is probably the initial step in the mechanism of microtubule formation.

Use of monoclonal antibodies to analyse the expression of a multi-tubulin family.

We have used a panel of monoclonal antibodies in a study of the expression of multiple tubulins in Physarum polycephalum. Three anti-beta-tubulin monoclonal antibodies, DM1B, DM3B3 and KMX-1 all reacted with the beta 1-tubulin isotypes expressed in both myxamoebae and plasmodia. However, these antibodies showed a spectrum of reduced reactivity with the plasmodial beta 2-tubulin isotype - the competence of recognition of this isotype was graded DM1B greater than KMX-1 greater than DM3B3. The anti-alpha-tubulin monoclonal antibody, YOL 1/34 defined the full complement of Physarum alpha-tubulin isotypes, whilst the anti-alpha-tubulin monoclonal antibody, KMP-1 showed a remarkably high degree of isotype specificity. KMP-1 recognises all of the myxamoebal alpha 1-tubulin isotypes but only recognises 3 out of the 4 alpha 1-tubulin isotypes expressed in the plasmodium (which normally focus in the same 2D gel spot). KMP-1 does not recognise the plasmodial specific alpha 2-tubulin isotype. This monoclonal antibody reveals a new level of complexity amongst the tubulin isotypes expressed in Physarum and suggests that monoclonal antibodies are valuable probes for individual members of multi-tubulin families.