Modifications to methods for the enumeration and detection of injured Escherichia coli O157:H7 in foods

Reliable methods are required for the detection and enumeration of potentially injured E. coli O157 in food in the presence of outnumbering competing bacteria. Selective agents can prevent or inhibit the recovery and subsequent multiplication of injured cells and direct inoculation, either into selective enrichment broths or onto selective agar plates is still used in many methods for E. coli O157 detection and enumeration. When compared with tryptone soya agar (TSA), sorbitol MacConkey agar (SMAC) was shown to underestimate the concentration of viable E. coli O157:H7 subjected to low pH and high NaCl concentration. Using a resuscitation stage on TSA followed by membrane transfer to SMAC improved recovery to levels obtained on TSA. The membrane method was used to monitor the numbers of artificially contaminated E. coli O157:H7 during the fermentation of a meat product and demonstrated better survival when compared to counts on SMAC. Six rapid methods for the detection of E. coli O157 in food (BAX E. coli O157, Reveal 8 E. coli O157-H7 screening test, VIP EHEC, VIDAS E. coli O157 (ECO), EHEC-Tek and Tecra E. coli O157 visual immunoassay), were evaluated using beetburgers, parsley and fermented meat artificially contaminated with injured cells. Methods using direct selective enrichment, with or without an elevated incubation temperature gave false-negative results. The incorporation of a non-selective pre-enrichment medium improved the detection rates of these assays by up to ten fold.     

Cleavage motifs of the yeast 20S proteasome beta subunits deduced from digests of enolase 1

The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active beta subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.     

Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast

The actin cytoskeleton in budding yeast consists of cortical patches and cables, both of which polarize toward regions of cell growth. Tropomyosin localizes specifically to actin cables and not cortical patches. Upon shifting cells with conditionally defective tropomyosin to restrictive temperatures, actin cables disappear within 1 min and both the unconventional class V myosin Myo2p and the secretory vesicle-associated Rab GTPase Sec4p depolarize rapidly. Bud growth ceases and the mother cell grows isotropically. When returned to permissive temperatures, tropomyosin-containing cables reform within 1 min in polarized arrays. Cable reassembly permits rapid enrichment of Myo2p at the focus of nascent cables as well as the Myo2p- dependent recruitment of Sec4p and the exocyst protein Sec8p, and the initiation of bud emergence. With the loss of actin cables, cortical patches slowly assume an isotropic distribution within the cell and will repolarize only after restoration of cables. Therefore, actin cables respond to polarity cues independently of the overall distribution of cortical patches and are able to directly target the Myo2p-dependent delivery of secretory vesicles and polarization of growth.     

Current and future therapies for HCV infection: what should the end point for treatment be?

Based upon all of the available data relating to the natural history, chemical course, and response to therapy of HCV, the following recommendations are made: 1) The primary end point for HCV therapy should be HCV clearance from all tissue sites, eg plasma, liver and others 2) Therapy should be provided for patients with early infections as they have the best chance of achieving a virologic response 3) Therapy should be offered to patients with cirrhotic disease, as prevention of hepatic decompensation and degeneration to hepatic cancer is possible 4) End stage decompensated disease should be treated, particularly if liver transplantation is being considered, in an effort to either eliminate or ameliorate disease recurrence 5) Combination therapies are preferable to monotherapy as they enhance the likelihood of a therapeutic response. Some of these include agents that reduce the frequency of IFN-induced untoward events (NSAIDs) 6) The approach to HCV infection should be to view it as an infectious disease. In this way, multi-agent therapy could be used to prevent the emergence of drug resistant mutants as well as to obtain earlier clearance of the infection than is possible with monotherapy.     

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.     

Analysis of fluoromethyl group chirality establishes a common stereochemical course for the enolpyruvyl transfers catalyzed by EPSP synthase and UDP-GlcNAc enolpyruvyl transferase

The stereochemistry of transient methyl group formation at C-3 of phosphoenolpyruvate (PEP) in the reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase has been examined using the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). Kinetically stable, chiral [1H, 2H]fluoromethyl analogs of the reaction tetrahedral intermediate were isolated and subjected to decomposition and stereochemical analysis. EPSP synthase was found to catalyze the 2-re face addition of solvent-derived hydrogen to C-3 of FPEP (corresponding to the 2-si face of PEP). Comparison of these data with prior analogous work on the MurA reaction [Kim, D.H., Lees, W.J., & Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381] suggests that the two enolpyruvyl transferases share a common stereochemical course, further strengthening the mechanistic, structural, and evolutionary relationship between the two enzymes.     

Comparative genomic hybridization: a comparison with molecular and cytogenetic analysis

A comparative analysis of the differentiation pattern, the proliferative behaviour, and the level of apoptosis between human benign and malignant neoplasms of smooth-muscle (SM) tissue is lacking. The clinical, histopathological, immunochemical, and immunocytochemical features of leiomyomas (LM) and leiomyosarcomas (LMS) were investigated by a panel of monoclonal antibodies specific for some differentiation markers of SM tissue (SM myosin and alpha-actin, desmin, and SM22) and for markers of non-muscle tissue (vimentin and non-muscle myosin). Proliferating normal and neoplastic cells were identified by proliferating-cell nuclear antigen (PCNA)/Ki67 immunostainings and the apoptotic cells were revealed by means of the terminal-deoxynucleotidyltransferase-mediated dUTP nick-end labelling technique. Gel electrophoresis and Western blotting, performed with anti-(SM1/SM2 myosin isoform) antibody, indicated quantitative differences between LMS and LM, which mirrored higher positive to negative nuclear ratios for PCNA, Ki67 and apoptosis in malignant as opposed to benign neoplasms. With LM, however, a similar SM1 to SM2 ratio could be associated with different proliferation levels. Uterine, gastric and intestinal LMS displayed specific patterns of SM1/SM2 and/or non-muscle myosin expression that were not paralleled by different levels of proliferation/apoptosis. While the level of PCNA/Ki67 correlated with the level of apoptosis in normal SM tissues and LM, that of LMS did not. In vivo at the cellular level, LM and uterine LMS displayed a near-uniform SM tissue differentiation, whereas the other LMS displayed a lesser or a heterogeneous immunoreactivity. In vitro, cultured LMS cells showed a limited and peculiar expression of SM myosin. In conclusion, there is no reciprocal relationship between degree of differentiation and the level of proliferation, as exemplified by the finding that the less differentiated intestinal LMS displays the lowest proliferative behaviour and that the relatively more differentiated gastric LMS/metastasis is more proliferative.     

Use of hospital services by AIDS patients with psychiatric illness

The purpose of this study was to assess the effect of psychiatric illness on length of stay and patterns of admission among AIDS patients hospitalized for medical illnesses. Medical records were abstracted for AIDS patients admitted to hospitals in Washington State from 1990 through 1992. Psychiatric comorbidity was defined by the presence of an International Classification of Disease-9 code reflecting psychiatric illness. Medical morbidity was addressed using CD4 count and AIDS-defining illnesses as markers of disease severity. Of 2834 admissions, 15% included one or more psychiatric diagnoses. Psychiatric illness (F 39.1; df 1,2830; p < 0.001) and discharge disposition (F 81.2; df 2,2830; p < 0.001) contributed significantly to the model, explaining increased length of stay (F 67.2; df 3,2830; p < 0.001). Future research needs to address the possible etiology of psychiatric comorbidity's contribution to length of stay and the effect on quality and cost of care.