In situ metal imaging and Zn ligand-speciation in a soil-dwelling sentinel: complementary electron microprobe and synchrotron microbeam X-ray analyses.

Understanding the relationships between accumulated metal speciation in cells and tissues of ecologically significant taxa such as earthworms will improve risk assessments. Synchrotron-based µ-focus X-ray spectroscopy was used to detect, localize, and determine ligand-speciation of Zn and Pb in thin sections of two epigeic earthworm species collected from a Pb/Zn-mine soil. The findings indicated that Zn and Pb partition predominantly as typical hard acids (i.e., strong affinities for O-donors) within liverlike chloragocytes. Moreover, Zn speciation was very similar in the chloragog and intestinal epithelia but differed subtly in the kidneylike nephridial tubules; neither Zn nor Pb was detectable in the ventral nerve cord. High resolution X-ray mapping of high pressure-frozen, ultrathin, freeze-substituted sections in a transmission electron microscope (TEM), combined with conventional TEM structural analysis, identified a new cell type packed with highly organized rough endoplasmic reticulum and containing deposits of Cd (codistributed with S); there was no evidence that these cells are major depositories of Zn or Pb. These data may be used in a systems biology approach to assist in the interpretation of metal-evoked perturbations in whole-worm transcriptome and metabolome profiles.

Metal ion trafficking in earthworms. Identification of a cadmium-specific metallothionein.

Exposure to cadmium poses a considerable risk to human health and environmental safety. Earthworms reside in the most contaminated sites on earth, displaying a phenomenal tolerance to toxic heavy metals. They exhibit a distinct metabolic pathway that allows the bio-accumulation of cadmium to yield body burdens in excess of 1/1000th of total dry body weight, a most impressive figure by any standard. However, the precise molecular mechanism underlying this phenomenon remains to be unraveled. This study meets this challenge by fully characterizing the major metal-binding protein in earthworms, namely the two isoforms of metallothionein. Chemical analysis of recombinant protein showed that although both isoforms bind equimolar amounts of cadmium (6 mol), wMT-2 is more stable during proton competition. Furthermore, isoform-specific transcript analysis demonstrated that only wMT-2 is responsive to cadmium in a dose and temporal manner. The specific sequestration of cadmium to wMT-2 protein was confirmed in situ using polyclonal antisera. The latter also provided the means for mapping the cellular and intracellular distribution of metallothionein, thus yielding a holistic insight into its involvement in cadmium transit during absorption, storage, and excretion. The structure-function relationship of wMT-2 and its role in cadmium detoxification through sequestration and compartmentalization is discussed.

Cl(-) channels in basolateral TAL membranes XV. Molecular heterogeneity between cortical and medullary channels.

We have isolated two new and highly homologous cDNAs, mmClC-Ka from mouse outer medulla and mcClC-Ka from mouse cortex. In both cases, mRNA was obtained from the indicated region and subjected to RT-PCR using primers from the nucleotide sequence of rbClC-Ka, which encodes basolateral Cl(-) channels (termed rbClC-Ka) in rabbit MTAL. The predicted protein products of mmClC-Ka and mcClC-Ka, mmClC-Ka and mcClC-Ka, respectively, were 85% homologous and had predicted molecular weights of 75 kDa. The predicted protein sequences for mmClC-Ka and rbClC-Ka had three cytosolic sites-threonine 185, threonine 187 and serine 270-which were absent in mcClC-Ka. These three moieties represent potential sites for phosphorylation of mmClC-Ka and rbClC-Ka, but not of mcClC-Ka, and may account for the failure of (ATP + PKA) to increase the open time probability P(o) in basolateral CTAL Cl(-) channels. We prepared antisense oligonucleotides specific for nonhomologous regions of these two cDNAs, mmAntisense for mmClC-Ka and mcAntisense for mcClC-Ka. Using anti-rbClC-Ka, a polyclonal antibody to rbClC-Ka, we found that, when transfected into cultured mouse MTAL and CTAL cells, mmAntisense suppressed the appearance of the 75 kDa band by 50% in vesicles from MTAL but not CTAL cells, while transfection of MTAL and CTAL cells with mcAntisense suppressed appearance of the 75 kDa band in vesicles from CTAL but not MTAL cells. mmAntisense transfection also prolonged the half-time (T(1/2), sec) for (36)Cl(-) efflux in cultured MTAL cells from 82.4 +/- 6.8 sec (sem) to 187.8 +/- 9.5 sec (n = 5; P = 0.0001) while mcAntisense transfection had no such effect. Conversely, in cultured CTAL cells, mcAntisense transfection prolonged the T(1/2) for (36)Cl(-) efflux from 80.9 +/- 6.3 sec to 191.8 +/- 6.5 sec (n = 5; P = 0.00005), while mmAntisense had no such effect. We conclude that mmClC-Ka and mcClC-Ka may encode the basolateral Cl(-) channels mediating net Cl(-) absorption in mouse MTAL and CTAL, respectively.

Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels.

The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.

Increased release of the N-terminus of the atrial natriuretic factor prohormone with increasing absolute atmospheres of pressure in a hyperbaric chamber and reversal with oxygen therapy.

Increasing atmospheres of absolute pressure (ATA) on the cardiopulmonary system results in a marked diuresis. The present investigation was designed to determine if the diuresis observed with increasing ATA is associated with increased release of the N-terminus of the atrial natriuretic factor (ANF) prohormone that contains two potent diuresis-producing hormones consisting of amino acids (aa) 1-30 (pro ANF 1-30; long-acting sodium stimulator) and aa 31-67 (pro ANF 31-67; vessel dilator) of this 126 aa prohormone. Seven healthy volunteers (mean age, 31 years) had the circulating concentration of the N-terminus of the ANF prohormone evaluated at 1, 2, and 3 ATA in a monoplace hyperbaric chamber by two specific and sensitive radioimmunoassays that immunologically recognize (1) the whole 98 aa N-terminus and (2) the midportion of the N-terminus consistent with aa 31-67 (pro ANF 31-67). With increasing ATA from 1 (sea level) to 2 (equivalent to 33 feet of sea water), the circulating concentrations of both the whole N-terminus and pro ANF 31-67 increased threefold. At 3 ATA (66 feet of sea water), their circulating concentrations increased sixfold over their concentrations, at 1 ATA. With the addition of 100 percent O2 while at 3 and 2 ATA, the circulating concentrations of both the whole N-terminus and pro ANF 31-67 immediately decreased to their prehyperbaric ATA levels and remained there with further decompression to 1 ATA and removal of O2 supplementation. The increased circulating concentration of the N-terminus of the ANF prohormone containing two peptides with potent diuretic effects during increasing atmospheres of absolute pressure may help to explain the diuresis that has been observed with increasing ATA.

Release of atrial natriuretic factor with increasing absolute atmospheres of pressure in a hyperbaric chamber and reversal with oxygen therapy.

Diving is associated with a marked diuresis which can be accurately reproduced in a hyperbaric chamber where the pressure of a specific depth can be precisely controlled. The present investigation was designed to determine if the diuresis observed with diving is associated with increased release of a new diuresis-producing hormone, atrial natriuretic factor (ANF). Seven experienced divers (3 men and 4 women; average age of 31 years) had their circulating concentration of atrial natriuretic factor measured at 1, 2 and 3 atmospheres of absolute pressure (ATA) in a monoplace hyperbaric chamber while breathing air. With the simulated dive to 33 feet (2 ATA), the circulating concentration of ANF increased 3-fold and at a depth of 66 feet (3 ATA) ANF increased 6-fold over the concentration at 1 ATA. With the addition of 100% oxygen while at depths of 33 and 66 feet, the concentration of ANF immediately decreased to its pre-dive concentration and remained there with further decompression to surface air and removal of oxygen supplementation. Thus, the increased release of ANF with depth may help to explain the diuresis observed with diving.

A study of patient acceptance of double-contrast barium enema and colonoscopy. Which procedure is preferred by patients?

To our knowledge, no previous study has addressed the question of which method of evaluation of the lower gastrointestinal tract is preferred by patients, air-contrast barium enema or colonoscopy. Over a four-month period, we asked 189 consecutive patients who had undergone colonoscopy to express their preference for either air-contrast barium enema or colonoscopy. A clear preference for colonoscopy was expressed by our patients in terms of comfort and polyp detection despite higher cost. Time lost from work and post-procedure constipation were significantly less for colonoscopy than for barium enema. These factors should be considered in the evaluation of suspected lower gastrointestinal tract disease.

The elemental composition of the chloragosomes of two earthworm species (Lumbricus terrestris and Allolobophora longa) determined by electron probe X-ray microanalysis of freeze-dried cryosections.

The elemental composition of the chloragosomes of the two earth-worm species, one with complex highly active Ca-secreting calciferous glands (L. terrestris) and the other with non-secretory glands (A. longa), was determined by the electron probe X-ray microanalysis of unfixed freeze-dried cryosections. The predominant constituents of the chloragosomes of both species were P, Ca, Zn and S, with lesser quantities of K, Cl and Fe. The most striking species differences in chloragosomal chemistry were the higher concentrations (expressed as relative mass fractions) of P(x 2.1), Ca(x 1.5), and Zn(x 2.3) in L. terrestris, and the much higher S(x 19.6) in A. longa. These differences were discussed in relation to the general ecophysiology of the two species, and more specifically in relation to heavy metal uptake and binding.