Isolation and Characterization of Polycyclic Aromatic Hydrocarbon–Degrading Mycobacterium Isolates from Soil

Bioremediation of soils contaminated with wood preservatives containing polycyclic aromatic hydrocarbons (PAHs) is desired because of their toxic, mutagenic, and carcinogenic properties. Creosote wood preservative–contaminated soils at the Champion International Superfund Site in Libby, Montana currently undergo bioremediation in a prepared-bed land treatment unit (LTU) process. Microbes isolated from these LTU soils rapidly mineralized the ¹⁴C-labeled PAH pyrene in the LTU soil. Gram staining, electron microscopy, and 16S rDNA-sequencing revealed that three of these bacteria, JLS, KMS, and MCS, were Mycobacterium strains. The phylogeny of the 16S rDNA showed that they were distinct from other Mycobacterium isolates with PAH-degrading activities. Catalase and superoxide dismutase (SOD) isozyme profiles confirmed that each isolate was distinct from each other and from the PAH-degrading mycobacterium, Mycobacterium vanbaalenii sp. nov, isolated from a petroleum-contaminated soil. We find that dioxygenase genes nidA and nidB are present in each of the Libby Mycobacterium isolates and are adjacent to each other in the sequence nidB-nidA, an order that is unique to the PAH-degrading mycobacteria.This revised version was published online in November 2004 with corrections to Volume 48.     

Covalent trimers of the internal N-terminal trimeric coiled-coil of gp41 and antibodies directed against them are potent inhibitors of HIV envelope-mediated cell fusion

We have engineered two soluble, covalently linked, trimeric polypeptides, N35CCG-N13 and N34CCG comprising only the internal trimeric coiled-coil of the ectodomain of HIV-1 gp41. Both trimers inhibit human immunodeficiency virus, type 1 (HIV-1) envelope (Env)-mediated cell fusion at nanomolar concentrations by targeting the exposed C-terminal region of the gp41 ectodomain in the prehairpin intermediate state. The IC50 values for N35CCG-N13 and N34CCG are approximately 15 and approximately 95 nM, respectively, in a quantitative vaccinia virus-based reporter gene assay for HIV-1 Env-mediated cell fusion using Env from the T cell tropic strain LAV. Polyclonal antibodies were raised against N35CCG-N13 and a tightly binding fraction of anti-N35CCG-N13 inhibits T cell and macrophage tropic HIV-1 Env-mediated cell fusion with respective IC50 values of approximately 0.5 and approximately 1.5 microg/ml at 37 degrees C. The tightly binding anti-N35CCG-N13 antibody fraction targets the exposed internal trimeric coiled-coil in the prehairpin intermediate state of gp41 in a manner analogous to peptides derived from the C region of the gp41 ectodomain. The potency of the tightly binding anti-N35CCG-N13 antibody fraction in the fusion assay is comparable with that of the broadly neutralizing monoclonal antibody 2G12. These results indicate that N35CCG-N13 is a potential anti-HIV therapeutic agent and represents a suitable immunogen for the generation of neutralizing monoclonal antibodies targeted to the internal trimeric coiled-coil of gp41. The data on the tightly binding anti-N35CCG-N13 antibody fraction demonstrate that the internal trimeric coiled-coil of gp41 in the prehairpin intermediate state is accessible to antibodies and that access is not restricted by either antibody size or the presence of a kinetic barrier.     

The effect of willow leaf extracts on human leukemic cells in vitro

The young developing leaves of willow (Salix safsaf, Salicaceae) trees have antileukemic activity. After a 24-h incubation in vitro, the crude water extracts of the leaves killed a majority of the blasts of acute myeloid leukemia (AML, 73.8%).     

Association of the X-linked lymphoproliferative disease gene product SAP/SH2D1A with 2B4, a natural killer cell-activating molecule, is dependent on phosphoinositide 3-kinase

Natural killer (NK) cells express an activating receptor, 2B4, that enhances cellular cytotoxicity. Upon NK cell activation by ligation of 2B4, the intracellular domain of 2B4 associates with the X-linked lymphoproliferative disease (XLP) gene product, signaling lymphocytic activation molecule-associated protein/SH2D1A (SAP/SH2D1A). Defective intracellular association of 2B4 with mutated SAP/SH2D1A is likely to underlie the defects in cytotoxicity observed in NK cells from patients with XLP. We report here a role for phosphoinositide 3-kinase (PI3K) in the recruitment and association of SAP/SH2D1A to 2B4 in human NK cells. The activation of normal NK cells by ligation of 2B4 leads to the phosphorylation of 2B4, recruitment of SAP/SH2D1A, and association of the p85 regulatory subunit of PI3K. The inhibition of PI3K enzymatic activity with either wortmannin or LY294002 prior to 2B4 ligation does not alter the association of 2B4 with the p85 subunit but prevents the recruitment of SAP/SH2D1A to 2B4. In addition, PI3K inhibitors significantly diminish the cytotoxic function of primary NK cells. This observed inhibition of cytotoxicity, present in normal NK cells, was less apparent or absent in NK cells derived from a patient with XLP. These data indicate that the cytotoxicity of activated NK cells is mediated by the association of 2B4 and SAP/SH2D1A, and that this association is dependent upon the activity of PI3K.     

Amyloidosis and periodic peritonitis (familial mediterranean Fever)

Some reports indicate that amyloidosis is a rare occurrence in persons with periodic peritonitis (familial Mediterranean fever), while others seem to show it occurs relatively frequently. Two cases were seen among 80 patients in Iraq. Twenty-one consecutive rectal biopsies were negative for amyloidosis. The variation in reported incidence is partly real and partly apparent. Amyloidosis occurs frequently in certain ethnic groups, and it is possible that there are two traits, one for periodic peritonitis and the other for amyloidosis.     

Effects of Calyculin A and Okadaic Acid on Acetylcholine Release and Subcellular Distribution in Rat Hippocampal Formation

Abstract : The mechanisms regulating the compartmentation of acetylcholine (ACh) and the relationship between transmitter release and ACh stores are not fully understood. In the present experiments, we investigated whether the inhibitors of serine/threonine phosphatases 1 and 2A, calyculin A and okadaic acid, alter subcellular distribution and the release of ACh in rat hippocampal slices. Calyculin A and okadaic acid significantly (p < 0.05) depleted the occluded ACh of the vesicular P₃ fraction, but cytoplasmic ACh contained in the S₃ fraction was not significantly affected. The P₃ fraction is known to be heterogeneous ; calyculin A and okadaic acid reduced significantly (p < 0.05) the amount of ACh recovered with a monodispersed fraction (D) of synaptic vesicles, but the other nerve terminal bound pools (E-F and G-H) were not so affected. K⁺-evoked ACh release decreased significantly (p < 0.01) in the presence of calyculin A and okadaic acid, suggesting that fraction D's vesicular store of ACh contributes to transmitter release. The loss of ACh from synaptic vesicle fractions prepared from tissue exposed to phosphatase inhibitors appeared not to result from a reduced ability to take up ACh. Thus, when tissue was allowed to synthesize [³H]ACh from [³H]choline, the ratio of [³H]ACh in the S₃ to P₃ fractions was not much changed by exposure of tissue to calyculin A or okadaic acid ; furthermore, the specific activity of ACh recovered from the D fraction was not reduced disproportionately to that of cytosolic ACh. The changes are considered to reflect reduced synthesis of ACh by tissue treated with the phosphatase inhibitors, rather than an effect on vesicle uptake mechanisms. Thus, exposure of tissue to calyculin A or okadaic acid appears to produce selective depletion of tissue ACh content in a subpopulation of synaptic vesicles, suggesting that phosphatases play a role in ACh compartmentation.     

Measurement of menadione in urine by HPLC

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C₃₀ column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H₂O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R²) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3 pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine.