Impact of Urban Pollution from the Hanoi Area on Benthic Diatom Communities Collected from the Red, Nhue and Tolich Rivers (Vietnam)

The effects of urban pollution from Hanoi city on the benthic diatom communities of the Nhue–Tolich river system were studied during the 2003 dry season. Benthic diatoms were allowed to grow on glass slides suspended in the water flow for 4 weeks. To reveal the relationship between water quality and diatom communities, Canonical correspondence analysis (CCA) was used on data concerning relative abundances of diatom species and environmental variables. Two diatom indices, IPS and DAIpo, were applied to evaluate water quality in the three rivers. A total of 291 diatom taxa were found in the Red, Nhue and Tolich Rivers. These were mainly cosmopolitan taxa, with some tropical, subtropical and endemic taxa. The most abundant taxa at the Red site were Aulacoseira granulata, Achnanthidium minutissimum, Encyonema minutum, Navicula recens and other halophilous taxa such as Nitzschia kurzii, Seminavis strigosa, Entomoneis paludosa, Bacillaria paradoxa. Diatom assemblages at the Tolich site consisted mainly of Nitzschia umbonata, Nitzschia palea and Eolimna minima. Diatom density ranged from 660 to 30,000 cells/cm². Environmental variables and diatom assemblage composition at all sites were significantly correlated. Two diatom indices gave similar results and indicate the Tolich River with the lowest values as a highly polluted site.     

Partial phase diagram of aqueous bovine carbonic anhydrase: analyses of the pressure-dependent temperatures of the low- to physiological-temperature nondenaturational conformational change and of unfolding to the molten globule state

At 1.0 atm pressure and in 150 mM sodium phosphate (pH = 7.0), bovine carbonic anhydrase undergoes a nondenaturational conformational change at 30.3 degrees C and an unfolding transition from the physiological conformer to the molten globule state at 67.4 degrees C. The pressure dependences of the temperatures of these transitions have been studied under reversible conditions for the purpose of understanding DeltaH degrees , DeltaS degrees , and DeltaV for each conformational change. Temperatures for the low-temperature to physiological-temperature conformational change T(L-->P) are obtained from physiologically relevant conditions using slow-scan-rate differential scanning calorimetry. Temperatures for the physiological-temperature conformation to molten globule state conversion T(P-->MG) are obtained from differential scanning calorimetry measurements of the apparent transition temperature in the presence of guanidine hydrochloride extrapolated to zero molar denaturant. The use of slow-scan-rate differential scanning calorimetry permits the calculation of the activation volume for the conversion of the low-temperature conformer to the physiological-temperature conformer DeltaV(double dagger)(L-->P). At 1.0 atm pressure, the transition from the low-temperature conformer to the physiological-temperature conformer involves a volume change DeltaV(L-->P) = 15 +/- 2 L/mole, which contrasts with the partial unfolding of the physiological-temperature conformer to the molten globule state (DeltaV(P-->MG) = 26 +/- 9 L/mole). The activation volume for this process DeltaV(double dagger)(L-->P) = 51 +/- 9 L/mole and is consistent with a prior thermodynamic analysis that suggests the conformational transition from the low-temperature conformation to the physiological-temperature conformation possesses a substantial unfolding quality. These results provide further evidence the structure of the enzyme obtained from crystals grown below 30 degrees C should not be regarded as the physiological structure (the normal bovine body temperature is 38.3 degrees C). These results should therefore have implications in any area that seeks to correlate the crystal structure of bovine carbonic anhydrase to physiological function.     

Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone

A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.     

Binding of myosin to actin in myofibrils during ATP hydrolysis

Measurements of cross-bridge attachment to actin in myofibrils during ATP hydrolysis require prior fixation of myofibrils to prevent their contraction. The optimal cross-linking of myofibrils was achieved by using 10 mM carbodiimide (EDC) under rigor conditions and at 4 degrees C. The fixed myofibrils had elevated MgATPase activity (150%) and could not contract. As judged by chymotryptic digestions and subsequent SDS gel electrophoresis analysis, less than 25% of myosin heads were cross-linked in these myofibrils. The isolated, un-cross-linked myosin heads showed pH-dependent Ca2+- and EDTA(K+)-ATPase activities similar to those of standard intact S-1. For measurements of myosin binding to actin, the modified myofibrils were digested with trypsin at a weight ratio of 1:50 under rigor, relaxed, and active-state conditions. Aliquots of tryptic digestion reactions were then cleaved with chymotrypsin to yield isolated myosin heads and their fragments. Analysis of the decay of myosin heavy-chain bands on SDS gels yielded the rates of myosin cleavage under all conditions and enabled the measurements of actomyosin binding in myofibrils in the presence of MgATP. Using this approach, we detected rigorlike binding of 25 +/- 6% of myosin heads to actin in myofibrils during ATP hydrolysis.     

Inhibition of alpha interferon signaling by hepatitis B virus

Alpha interferon (IFN-alpha) and pegylated IFN-alpha (pegIFN-alpha) are used for the treatment of chronic hepatitis B (CHB). Unfortunately, only a minority of patients can be cured. The mechanisms responsible for hepatitis B virus (HBV) resistance to pegIFN-alpha treatment are not known. pegIFN-alpha is also used to treat patients with chronic hepatitis C (CHC). As with chronic hepatitis B, many patients with chronic hepatitis C cannot be cured. In CHC, IFN-alpha signaling has been found to be inhibited by an upregulation of protein phosphatase 2A (PP2A). PP2A inhibits protein arginine methyltransferase 1 (PRMT1), the enzyme that catalyzes the methylation of the important IFN-alpha signal transducer STAT1. Hypomethylated STAT1 is less active because it is bound by its inhibitor, PIAS1. In the present work, we investigated whether similar molecular mechanisms are also responsible for the IFN-alpha resistance found in many patients with chronic hepatitis B. We analyzed the expression of PP2A, the enzymatic activity of PRMT1 (methylation assays), the phosphorylation and methylation of STAT1, the association of STAT1 with PIAS1 (via coimmunoprecipitation assays), the binding of activated STAT1 to interferon-stimulated response elements (via electrophoretic mobility shift assays), and the induction of interferon target genes (via real-time RT-PCR) in human hepatoma cells expressing HBV proteins as well as in liver biopsies from patients with chronic hepatitis B and from controls. We found an increased expression of PP2A and an inhibition of IFN-alpha signaling in cells expressing HBV proteins and in liver biopsies of patients with CHB. The molecular mechanisms involved are similar to those found in chronic hepatitis C.     

The AprX protein of Pseudomonas aeruginosa: a new substrate for the Apr type I secretion system

Protein secretion in Pseudomonas aeruginosa involves different mechanisms. The type II and type III secretory pathways control the extracellular release of a wide range of substrates. The type I secretion process, or ABC transporter, was believed to be exclusively involved in alkaline protease secretion. Recently, it was discovered that a P. aeruginosa heme binding protein, HasAp, is also secreted by a type I process. We present here the identification of a third putative type I-dependent protein of P. aeruginosa, AprX. The function of this protein has not yet been elucidated but very interestingly it appears to be linked to the apr cluster, and organized in one single operon together with the aprD, -E and -F genes.