A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

F ricker , C.R. 1984. A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium. Journal of Applied Bacteriology 56 , 305–309.
The efficiency of Rappaport's broth (RB10) and Rappaport's broth containing novobiocin (NRB10) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used; no significant difference in their ability to recover salmonellae was demonstrated.     

Comparative study of the regeneration of the microcirculation of rabbit femur bone marrow after curetage and at the time of experimental myelosclerosis]

Destruction and regeneration of the bone marrow microcirculation in rabbit femur have been studied after curetage and experimental myelosclerosis. Different lesions lead to comparable restoration processes such as: installation of supplying vascular networks, appearance of a neovascularisation progressively replaced by neighboring structures of normal interadipocytic networks.     

Homologies in Cambrian Onychophora

Marine animals related to Recent onychophorans form a significant component in Cambrian faunas. Twelve characters are analysed for homologies in the seven best known Cambrian onychophorans. New morphological evidence and homology analyses for several characters indicate an anteroposterior reversal of Hallucigenia and Microdictyon . Proposed expansion of the trunk in Microdietyon during compaction is rejected. A jaw is tentatively identified in Onychodictyon . The shape of the annulations and the disposition of the tenth leg pair in Aysheaia are reinterpreted, and the suggestion of two somites to the first appendage pair is rejected. A suggested morphocline may mirror the phylogeny of the group. The taxonomic confusion surrounding the supposed radiolarian family Eoconchariidae is cleared     

Localization of metallocarboxypeptidase D in AtT-20 cells. Potential role in prohormone processing.

Carboxypeptidase D (CPD) is a recently discovered metallocarboxypeptidase that is predominantly located in the trans-Golgi network (TGN), and also cycles between the cell surface and the TGN. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. CPD-containing compartments were isolated using antibodies to the CPD cytosolic tail. The immunopurified vesicles contained TGN proteins (TGN38, furin, syntaxin 6) but not lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that CPD is present within the TGN and immature secretory granules but is virtually absent from mature granules, suggesting that CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the present study is that a soluble truncated form of CPD is secreted mainly via the constitutive pathway in AtT-20 cells, indicating that the lumenal domain does not contain signals for the sorting of CPD to mature secretory granules. Taken together, these data are consistent with the proposal that CPD participates in the processing of proteins within the TGN and immature secretory vesicles.     

Biosynthesis and packaging of carboxypeptidase D into nascent secretory vesicles in pituitary cell lines.

Metallocarboxypeptidase D (CPD) is a membrane-bound trans-Golgi network (TGN) protein. In AtT-20 cells, CPD is initially produced as a 170-kDa endoglycosidase H-sensitive glycoprotein. Within 30 min of chase, the CPD increases to 180 kDa and is resistant to endoglycosidase H as a result of carbohydrate maturation. CPD also undergoes an activation step required for binding to a substrate affinity resin. Blocking the protein exit from the endoplasmic reticulum inhibits the increase in molecular mass but not the step required for affinity column binding, suggesting that enzyme activation precedes carbohydrate maturation and that these reactions occur in distinct intracellular compartments. Only the higher molecular weight mature CPD enters nascent secretory vesicles, which bud from the TGN of permeabilized AtT-20 and GH3 cells. The budding efficiency of CPD into vesicles is 2-3-fold lower than that of endogenous proopiomelanocortin in AtT-20 cells or prolactin in GH3 cells. In contrast, the packaging of a truncated form of CPD, which lacks the cytoplasmic tail and transmembrane domain, was similar to that of proopiomelanocortin. Taken together, the results support the proposal that CPD functions in the TGN in the processing of proteins that transit the secretory pathway and that the C-terminal region plays a major role in TGN retention.     

Neuropeptide processing profile in mice lacking prohormone convertase-1

Prohormone convertase 1 (PC1; also known as PC3) is believed to be responsible for the processing of many neuropeptide precursors. To look at the role PC1 plays in neuropeptide processing in brain and pituitary, we used radioimmunoassays (RIA) as well as quantitative peptidomic methods and examined changes in the levels of multiple neuropeptide products in PC1 knockout (KO) mice. The processing of proenkephalin was impaired in PC1 KO mouse brains with a decrease in the level of Met-Enkephalin immunoreactivity (ir-Met-Enk) and an accumulation of higher molecular weight processing intermediates containing ir-Met-Enk. Processing of the neuropeptide precursor VGF was also affected in PC1 KO mouse brains with a decrease in the level of an endogenous 3 kDa C-terminal peptide. In contrast, the processing of proSAAS into PEN was not altered in PC1 KO mouse brains. Quantitative mass spectrometry was used to analyze a number of peptides derived from proopiomelanocortin (POMC), provasopressin, prooxytocin, chromogranin A, chromogranin B, and secretogranin II. Among them, the levels of oxytocin and peptides derived from chromogranin A and B dramatically decreased in the PC1 KO mouse pituitaries, while the levels of peptides derived from proopiomelanocortin and provasopressin did not show substantial changes. In conclusion, these results support the notion that PC1 plays a key role in the processing of multiple neuroendocrine peptide precursors and also reveal the presence of a redundant system in the processing of a number of physiologically important bioactive peptides.     

Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4 degrees C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4',6'-diamidino-2-phenylindole-propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable alternative viability surrogates are identified for disinfection studies.     

Substituting c-Jun N-terminal kinase-3 (JNK3) ATP-binding site amino acid residues with their p38 counterparts affects binding of JNK- and p38-selective inhibitors

c-Jun N-terminal kinase (JNK) activation is linked to the aberrant cell death in several neurodegenerative disorders, including Parkinson's and Alzheimer's disease. The sequence similarity among the JNK isoforms and fellow MAP kinase family member p38 has rendered the challenge of producing JNK3-specific inhibitors difficult. Using the crystal structure of JNK3 complexed with JNK inhibitors, potential compound-interacting amino acid residues were mutated to the corresponding residues in p38. The effects of these mutations on the kinetic parameters with three compounds were examined: a JNK3- (vs. p38-) selective inhibitor (SP 600125); a p38-selective inhibitor (Merck Z); and a potent combined JNK3 and p38 inhibitor (Merck Y). The data confirm the role of the JNK3 residues Ile-70 and Val-196 in both inhibitor and ATP-binding. Remarkably, the Ile-70-Val and Val-196-Ala mutations caused an increase and decrease, respectively, in the binding affinity of the p38-specific compound, Merck Z, of 10-fold. The Ile-70-Val effect may be due to the increased capacity of the active site to accommodate Merck Z, whereas the Val-196-Ala mutant may induce an unfavourable conformational change. Conservative mutations of the Asn-152 and Gln-155 residues inactivated the JNK3 enzyme, possibly interfering with protein folding in a critical hinge region of the protein.     

Peptidomics of Cpe fat/fat mouse hypothalamus: effect of food deprivation and exercise on peptide levels

Carboxypeptidase E is a major enzyme in the biosynthesis of numerous neuroendocrine peptides. Previously, we developed a technique for the isolation of neuropeptide-processing intermediates from mice that lack carboxypeptidase E activity (Cpe fat/fat mice) due to a naturally occurring point mutation. In the present study, we used a differential labeling procedure with stable isotopic tags and mass spectrometry to quantitate the relative changes in a number of hypothalamic peptides in Cpe fat/fat mice in two different paradigms that each cause an approximately 10% decrease in body mass. One paradigm involved a 2-day fast under normal sedentary conditions (i.e. standard mouse cages); the other involved giving mice access to an exercise wheel for 4 weeks with free access to food. Approximately 50 peptides were detected in both studies, and over 80 peptides were detected in at least one of the two studies. Twenty-eight peptides were increased >50% by food deprivation, and some of these were increased by 2- to 3-fold. In contrast, only three peptides were increased >50% in the group with exercise wheels, and many peptides showed a slight 15-30% decrease upon exercise. Approximately one-half of the peptides detected in both studies were identified by tandem mass spectrometry. Peptides found to be elevated by food deprivation but not exercise included a number of fragments of proenkephalin, prothyrotropin-releasing hormone, secretogranin II, chromogranin B, and pro-SAAS. Taken together, the differential regulation of these peptides in the two paradigms suggests that the regulation is not due to the lower body weight but to the manner in which the paradigms achieved this lower body weight.     

Quantitative in vivo measurement of glutathione in Arabidopsis cells

A new, non-destructive assay is described to quantify cytoplasmic glutathione (GSH) levels in vivo in single cells or populations of cells from Arabidopsis suspension cultures. Cytoplasmic GSH was labelled with monochlorobimane (MCB) in situ to give a fluorescent GSH-bimane (GSB) conjugate. At low (10-100 microM) concentrations of MCB, labelling was mediated by a glutathione S-transferase, which confers specificity for GSH. HPLC analysis of MCB-labelled low molecular-weight thiols showed that the assay measures the total GSH pool, including the oxidized glutathione. The progress curve for the labelling could be described using Michaelis-Menten kinetics with an apparent KM of 40 microM and Vmax of 470 micromol lcyt -1 min-1. There was no evidence for de novo synthesis of GSH during the labelling period of 2 h, suggesting that control of GSH synthesis is not mediated by feedback control of gamma-glutamylcysteine synthetase in this system. The total cellular level of GSH was calculated from the plateau value of the progress curve, after appropriate calibration, as 830-942 nmol g-1 FW. The volume fraction of cytoplasm was measured from serial optical sections of bimane-labelled cells collected by confocal laser scanning microscopy (CLSM) with excitation 442 nm, or two-photon laser scanning microscopy (TPLSM) with excitation 770 nm. A value of 42 +/- 3% cytoplasm was determined by manual segmentation, and a value of 37 +/- 2% using stereological techniques. Using these figures, values for cytoplasmic [GSH] were estimated to be between 2.7 +/- 0.3 and 3.2 +/- 0.3 mM for cell populations. In addition, measurement of GSH levels in individual cells using CLSM and TPLSM gave values of 3.0 +/- 0.5 and 3.5 +/- 0.7 mM, respectively.