Protein organization in mouse liver peroxisomes.

Peroxisomes from mouse liver were fractionated with Triton X-114, a procedure which yields a detergent phase consisting of proteins containing hydrophobic binding sites, and a nondetergent, or aqueous, phase containing hydrophilic proteins. When this method was applied to peroxisomes from control mice, catalase and fatty acyl-CoA oxidase distributed to the aqueous phase, whereas the integral membrane protein, PMP68, and the bifunctional protein were recovered exclusively in the detergent phase. Urate oxidase distributed intermediate between these two phases. With peroxisomes from mice treated with the peroxisome proliferator clofibrate, the bifunctional protein was recovered in both the detergent and the aqueous phases, and urate oxidase was shifted toward the aqueous phase. Other analyses of the subperoxisomal distribution of the bifunctional protein were consistent with a proportion of this protein being tightly associated with the peroxisomal membrane, or with some other uncharacterized, poorly soluble, component. Sucrose gradient centrifugation of the aqueous phase resulting from Triton X-114 fractionation of peroxisomes revealed that a major proportion of catalase, fatty acyl-CoA oxidase, the bifunctional protein, and other unidentified proteins behaved as if associated under these conditions. In this respect, use of a higher concentration of Triton X-114 for peroxisome fractionation led to the partitioning of some catalase and fatty acyl-CoA oxidase to the detergent phase, indicating the presence of some detergent-accessible hydrophobic binding sites even on these proteins. These data have been interpreted as indicating matrix protein associations in vivo, associations which may be responsive to proliferator treatment.     

Extraction of fluorescent cell puncta by adaptive fuzzy segmentation

MOTIVATION: The discrimination and measurement of fluorescent-labeled vesicles using microscopic analysis of fixed cells presents a challenge for biologists interested in quantifying the abundance, size and distribution of such vesicles in normal and abnormal cellular situations. In the specific application reported here, we were interested in quantifying changes to the population of a major organelle, the peroxisome, in cells from normal control patients and from patients with a defect in peroxisome biogenesis. In the latter, peroxisomes are present as larger vesicular structures with a more restricted cytoplasmic distribution. Existing image processing methods for extracting fluorescent cell puncta do not provide useful results and therefore, there is a need to develop some new approaches for dealing with such a task effectively. RESULTS: We present an effective implementation of the fuzzy c-means algorithm for extracting puncta (spots), representing fluorescent-labeled peroxisomes, which are subject to low contrast. We make use of the quadtree partition to enhance the fuzzy c-means based segmentation and to disregard regions which contain no target objects (peroxisomes) in order to minimize considerable time taken by the iterative process of the fuzzy c-means algorithm. We finally isolate touching peroxisomes by an aspect-ratio criterion. The proposed approach has been applied to extract peroxisomes contained in several sets of color images and the results are superior to those obtained from a number of standard techniques for spot extraction. AVAILABILITY: Image data and computer codes written in Matlab are available upon request from the first author.     

Osmosensor ProP of Escherichia coli responds to the concentration,chemistry, and molecular size of osmolytes in the proteoliposome lumen

Transporter ProP of Escherichia coli mediates the cellular accumulation of organic zwitterions in response to increased extracellular osmolality. We compared and characterized the osmoregulation of ProP activity in cells and proteoliposomes to define the osmotic shift-induced cellular change(s) to which ProP responds. ProP-(His)(6) activity in cells and proteoliposomes was correlated with medium osmolality, not osmotic shift, turgor pressure, or membrane strain. Both K(M) and V(max) for proline uptake via ProP-(His)(6) increased with increasing medium osmolality, as would be expected if osmolality controls the proportions of transporter with inactive and active conformations. The osmolality yielding half-maximal ProP-(His)(6) activity was higher in proteoliposomes than in cells. The osmolality response of ProP is also attenuated in bacteria lacking soluble protein ProQ. Indeed, the catalytic constant (k(cat)) for ProP-(His)(6) in proteoliposomes approximated that of ProP in intact bacteria lacking ProQ. Thus, the proteoliposome system may replicate a primary osmosensory response that can be further amplified by ProQ. ProP-(His)(6) is designated as an osmosensor because its activity is dependent on the osmolality, but not the composition, of the assay medium to which the cell surface is exposed. In contrast, ProP-(His)(6) activity was dependent on both the osmolality and the composition of the lumen in osmolyte-loaded proteoliposomes. For proteoliposomes containing inorganic salts, glucose, or poly(ethylene glycol) 503, transporter activity correlated with total lumenal cation concentration. In contrast, for proteoliposomes loaded with larger poly(ethylene glycol)s, the osmolality, the lumenal cation concentration, and the lumenal ionic strength at half-maximal transporter activity decreased systematically with poly(ethylene glycol) radius of gyration (range 0.8-1.8 nm). These data suggest that ProP-(His)(6) responds to osmotically induced changes in both cytoplasmic K(+) levels and the concentration of cytoplasmic macromolecules.     

Novel role for low molecular weight plasma thiols in nitric oxide-mediated control of platelet function

Nitric oxide (NO) is a powerful antiplatelet agent, but its notoriously short biological half-life limits its potential to prevent the activation of circulating platelets. Here we used diethylamine diazeniumdiolate (DEA/NO) as an NO generator to determine whether the antiplatelet effects of NO are prolonged by the formation of a durable, plasma-borne S-nitrosothiol reservoir. Preincubation of both platelet rich plasma (PRP) and washed platelets (WP) with DEA/NO (2 microm) for 1 min inhibited collagen-induced platelet aggregation by 82 +/- 5 and 91 +/- 2%, respectively. After 30 min preincubation with DEA/NO, NO was no longer detectable in either preparation, but aggregation remained markedly inhibited (72 +/- 7%) in PRP. In contrast, the inhibitory effect in WP was almost completely lost at this time (5 +/- 3%) but was partially restored (39 +/- 10%) in WP containing human serum albumin (1%) and fully restored by co-incubation with albumin and the low molecular weight (LMW) thiols, glutathione, (5 microm), cysteinyl-glycine (10 microm), or cysteine (10 microm). This NO-mediated effect was not seen with LMW thiols in the absence of albumin and was associated with S-nitrosothiol formation. Our results demonstrate that LMW thiols play an important role in both the formation and activation of an S-nitrosoalbumin reservoir that significantly prolongs the duration of action of NO.     

"No practical capabilities": American biological and chemical warfare programs during the Korean war

Much controversy still surrounds accusations that American forces in the Far East during the Korean War used biological warfare against North Korea and China. An analysis of recently declassified documents reveals that, although the United States attempted to accelerate its development and acquisition of such weapons during that period, its efforts to create a viable biological warfare capability were unsuccessful. Plans to similarly expand chemical warfare stocks and capabilities were also frustrated. Technological difficulties, personnel shortages, bureaucratic battles between the armed services, and policy limitations combined to hold back advances in American chemical and biological warfare. In light of the recent fears of terrorist attacks with such weapons, this analysis highlights the great difficulties involved in developing, acquiring, and delivering such capabilities.     

Purification and characterization of a doxorubicin-inhibited NADH-quinone (NADH-ferricyanide) reductase from rat liver plasma membranes

Plasma membrane-associated redox systems play important roles in regulation of cell growth, internal pH, signal transduction, apoptosis, and defense against pathogens. Stimulation of cell growth and stimulation of the redox system of plasma membranes are correlated. When cell growth is inhibited by antitumor agents such as doxorubicin, capsaicin, and antitumor sulfonylureas, redox activities of the plasma membrane also are inhibited. A doxorubicin-inhibited NADH-quinone reductase was characterized and purified from plasma membranes of rat liver. First, an NADH-cytochrome b(5) reductase, which was doxorubicin-insensitive, was removed from the plasma membranes by the lysosomal protease, cathepsin D. After removal of the NADH-cytochrome b(5) reductase, the plasma membranes retained a doxorubicin-inhibited NADH-quinone reductase activity. The enzyme, with an apparent molecular mass of 57 kDa, was purified 200-fold over the cathepsin D-treated plasma membranes. The purified enzyme had also an NADH-coenzyme Q(0) reductase (NADH: external acceptor (quinone) reductase; EC 1.6.5.) activity. Partial amino acid sequence of the enzyme showed that it was unique with no sequence homology to any known protein. Antibody against the enzyme (peptide sequence) was produced and affinity-purified. The purified antibody immunoprecipitated both the NADH-ferricyanide reductase activity and NADH-coenzyme Q(0) reductase activity of plasma membranes and cross-reacted with human chronic myelogenous leukemia K562 cells and doxorubicin-resistant human chronic myelogenous leukemia K562R cells. Localization by fluorescence microscopy showed that the reaction was with the external surface of the plasma membranes. The doxorubicin-inhibited NADH-quinone reductase may provide a target for the anthracycline antitumor agents and a candidate ferricyanide reductase for plasma membrane electron transport.     

PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification

In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.