The Pseudomonas putida strain SP1 was isolated from marine environment and was found to be resistant to 280 μM HgCl₂. SP1 was also highly resistant to other metals, including CdCl₂, CoCl₂, CrCl₃, CuCl₂, PbCl₂, and ZnSO₄, and the antibiotics ampicillin (Ap), kanamycin (Kn), chloramphenicol (Cm), and tetracycline (Tc). mer operon, possessed by most mercury-resistant bacteria, and other diverse types of resistant determinants were all located on the bacterial chromosome. Cold vapor atomic absorption spectrometry and a volatilization test indicated that the isolated P. putida SP1 was able to volatilize almost 100% of the total mercury it was exposed to and could potentially be used for bioremediation in marine environments. The optimal pH for the growth of P. putida SP1 in the presence of HgCl₂ and the removal of HgCl₂ by P. putida SP1 was between 8.0 and 9.0, whereas the optimal pH for the expression of merA, the mercuric reductase enzyme in mer operon that reduces reactive Hg²⁺ to volatile and relatively inert monoatomic Hg⁰ vapor, was around 5.0. LD₅₀ of P. putida SP1 to flounder and turbot was 1.5 × 10⁹ CFU. Biofilm developed by P. putida SP1 was 1- to 3-fold lower than biofilm developed by an aquatic pathogen Pseudomonas fluorescens TSS. The results of this study indicate that P. putida SP1 is a low virulence strain that can potentially be applied in the bioremediation of HgCl₂ contamination over a broad range of pH.

Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions. Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane water channels.

The caspases are a family of at least 10 human cysteine proteases that participate in cytokine maturation and in apoptotic signal transduction and execution mechanisms. Peptidic inhibitors of these enzymes are capable of blocking cytokine maturation and apoptosis, demonstrating their crucial roles in these processes. We have recently discovered that nitric oxide (NO), produced either extracellularly by NO donors or intracellularly by the inducible nitric oxide synthase, prevented apoptosis in hepatocytes. Caspase-3-like activity was found to be inhibited under these conditions. To investigate further the interaction between NO and caspases, we utilized purified human recombinant caspases and examined the effect of NO on enzymatic activities of different caspases. We report here that of the seven caspases studied, all were reversibly inhibited by NO. Dithiothreitol was able to reverse the NO inhibition, indicating direct S-nitrosylation of caspase catalytic cysteine residue by NO. Our results support the concept that NO is an endogenous regulator of caspase activity.

Water channels provide the plasma membranes of red cells and renal proximal tubules with high permeability to water, thereby permitting water to move in the direction of an osmotic gradient. Molecular identification of CHIP28 protein as the membrane water channel was first accomplished by measurement of osmotic swelling of Xenopus oocytes injected with CHIP28 RNA (Preston, G.M., Carroll, T.P., Guggino, W.B., and Agre, P.(1992) Science 256, 385-387). Since water channels are pharmacologically inhibited by submillimolar concentrations of Hg2+, site-directed mutagenesis was undertaken to demonstrate which of the 4 cysteines (87, 102, 152, or 189) is the Hg(2+)-sensitive residue in the CHIP28 molecule. Each cysteine was individually replaced by serine, and oocytes expressing each of the four mutants exhibited osmotic water permeability (Pf) equivalent to wild-type CHIP28. After incubation in HgCl2, all were significantly inhibited, except C189S exists as a multisubunit complex in the native membrane; however, although oocytes injected with mixed CHIP28 and C189S RNAs exhibited Pf corresponding to the sum of their individual activities, exposure to Hg2+ only reduced the Pf to the level of the C189S mutant. Of the six substitutions at residue 189, only the serine and alanine mutants exhibited increased Pf and had glycosylation patterns resembling wild-type CHIP28 on immunoblots. These studies demonstrated:(i) CHIP28 water channel activity is retained despite substitution of individual cysteines with serine;(ii) cysteine 189 is the Hg(2+)-sensitive residue;(iii) the subunits of the CHIP28 complex are individually active water pores;(iv) residue 189 is critical to proper processing of the CHIP28 protein.

Reconstitution of highly purified aquaporin CHIP (channel-forming integral protein) into proteoliposomes was previously shown to confer high osmotic water permeability (Pf) to the membranes [Zeidel et al.(1992) Biochemistry 31, 7436-7440]. Here we report detailed ultrastructural, pharmacologic, and transport studies of human red cell CHIP in proteoliposomes. Freeze-fracture and transmission electron microscopy revealed a uniform distribution of CHIP which was incorporated into the membranes in both native and inverse orientations. Morphometric analysis of membranes reconstituted at three different concentrations of CHIP revealed that the intramembrane particles correspond to tetramers or possible higher order oligomers, and the Pf increased in direct proportion to the CHIP density. Proteolytic removal of the 4-kDa C-terminal cytoplasmic domain of CHIP did not alter the Pf or oligomerization in red cell membranes. CHIP exhibited a similar conductance for water when reconstituted into membranes of varied lipid compositions. The sensitivities of CHIP-mediated Pf to specific sulfhydryl reagents were identical to known sensitivities of red cell Pf, including a delayed response to p-(chloromercuri)benzenesulfonate. CHIP did not increase the permeability of the proteoliposome membranes to H+/OH- or NH3. These studies demonstrate that CHIP proteoliposomes exhibit all known characteristics of water channels in native red cells and therefore provide a defined system for biophysical analysis of transmembrane water movements.

Aquaporin 1, a six-transmembrane domain protein, is a water channel present in many fluid-secreting and -absorbing cells. In Xenopus oocytes injected with aquaporin 1 complementary RNA, the application of forskolin or cyclic 8-bromo- adenosine 3',5'-monophosphate increased membrane permeability to water and triggered a cationic conductance. The cationic conductance was also induced by direct injection of protein kinase A (PKA) catalytic subunit, reduced by the kinase inhibitor H7, and blocked by HgCl2, an inhibitor of aquaporin 1. The cationic permeability of the aquaporin 1 channel is activated by a cyclic adenosine monophosphate-dependent mechanism that may involve direct or indirect phosphorylation by PKA.

Cholangiocytes line the intrahepatic bile ducts and regulate salt and water secretion during bile formation, but the mechanism(s) regulating ductal water movement remains obscure. A water-selective channel, the aquaporin CHIP, was recently described in several epithelia, so we tested the hypothesis that osmotic water movement by cholangiocytes is mediated by CHIP. Isolated rodent cholangiocytes showed a rapid increase in volume in the presence of hypotonic extracellular buffers; the ratio of osmotic to diffusional permeability coefficients was > 10. The osmotically induced increase in cholangiocyte volume was inversely proportional to buffer osmolality, independent of temperature, and reversibly blocked by HgCl2. Also, the luminal area of isolated, enclosed bile duct units increased after exposure to hypotonic buffer and was reversibly inhibited by HgCl2. RNase protection assays, anti-CHIP immunoblots, and immunocytochemistry confirmed that CHIP transcript and protein were present in isolated cholangiocytes but not in hepatocytes. These results demonstrate that (i) isolated cholangiocytes and intact, polarized bile duct units manifest rapid, mercury-sensitive increases in cell size and luminal area, respectively, in response to osmotic gradients and (ii) isolated cholangiocytes express aquaporin CHIP at both the mRNA and the protein level. The data implicate aquaporin water channels in the transcellular movement of water across cholangiocytes lining intrahepatic bile ducts and provide a plausible molecular explanation for ductal water secretion.

Previous studies have established that in susceptible mouse strains, such as A.SW (H-2s), repeated injections of subtoxic doses of HgCl2 induce increased serum levels of total IgE and IgG1, high serum titers of antinuclear autoantibodies (ANo1A), and immune-complex glomerulonephritis. Moreover, it has been shown that susceptibility is determined by H-2As and that Th cells are required for the induction of these immunopathologic alterations by HgCl2. In the present study we showed that treatment in vivo with anti-IL-4 mAb completely abrogated the HgCl2-induced increase in total IgE and partially inhibited the increase in IgG1, but failed to suppress the increase in IgG2A. Furthermore, we showed that IL-4 influences the pattern of IgG subclass distribution among ANo1A of HgCl2-treated mice. Whereas treatment with anti-IL-4 mAb significantly reduced the titers of IgG1 ANolA, it increased those of IgG2A, IgG2B, and IgG3 ANolA. Thus, these results show that IL-4 contributes to the optimal formation in vivo of murine IgG1 and that it is involved in the autoantibody formation of a systemic autoimmune disease. The available evidence suggests that HgCl2 induces an increased production of IL-4 by Th2 cells. If this is correct, it implies that MHC class II alleles determine whether the preferential response to HgCl2 is made by Th1 or Th2 cells and, hence, the type of immunopathologic alterations ensuing.

Plasmid pJP4 is an 80-kilobase, IncP1, broad-host-range conjugative plasmid of Alcaligenes eutrophus encoding resistance to mercuric chloride and phenyl mercury acetate and degradation of 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoate. By the use of cloning, transposon mutagenesis, and restriction endonuclease analysis, a biophysical and genetic map of pJP4 was generated.

Adipose tissue is a major site of glycerol production in response to energy balance. However, molecular basis of glycerol release from adipocytes has not yet been elucidated. We recently cloned a novel member of the aquaporin family, aquaporin adipose (AQPap), which has glycerol permeability. The current study was designed to examine the hypothesis that AQPap serves as a glycerol channel in adipocytes. Adipose tissue expressed AQPap mRNA in high abundance, but not the mRNAs for the other aquaglyceroporins, AQP3 and AQP9, indicating that AQPap is the only known aquaglyceroporin expressed in adipose tissue. Glycerol release from 3T3-L1 cells was increased during differentiation in parallel with AQPap mRNA levels and suppressed by mercury ion, which inhibits the function of AQPs, supporting AQPap functions as a glycerol channel in adipocytes. Fasting increased and refeeding suppressed adipose AQPap mRNA levels in accordance with plasma glycerol levels and oppositely to plasma insulin levels in mice. Insulin dose-dependently suppressed AQPap mRNA expression in 3T3-L1 cells. AQPap mRNA levels and adipose glycerol concentrations measured by the microdialysis technique were increased in obese mice with insulin resistance. Accordingly, negative regulation of AQPap expression by insulin was impaired in the insulin-resistant state. Exposure of epinephrine translocated AQPap protein from perinuclear cytoplasm to the plasma membrane in 3T3-L1 adipocytes. These results strongly suggest that AQPap plays an important role in glycerol release from adipocytes.

Leaf-moving organs, remarkable for the rhythmic volume changes of their motor cells, served as a model system in which to study the regulation of membrane water fluxes. Two plasma membrane intrinsic protein homolog genes, SsAQP1 and SsAQP2, were cloned from these organs and characterized as aquaporins in Xenopus laevis oocytes. Osmotic water permeability (P(f)) was 10 times higher in SsAQP2-expressing oocytes than in SsAQP1-expressing oocytes. SsAQP1 was found to be glycerol permeable, and SsAQP2 was inhibited by 0.5 mM HgCl(2) and by 1 mM phloretin. The aquaporin mRNA levels differed in their spatial distribution in the leaf and were regulated diurnally in phase with leaflet movements. Additionally, SsAQP2 transcription was under circadian control. The P(f) of motor cell protoplasts was regulated diurnally as well: the morning and/or evening P(f) increases were inhibited by 50 microM HgCl(2), by 2 mM cycloheximide, and by 250 microM phloretin to the noon P(f) level. Our results link SsAQP2 to the physiological function of rhythmic cell volume changes.