Diffuse adherence, ST-I enterotoxin and CFA/IV colonization factor are encoded by the same plasmid in the Escherichia coli O29:H21 strain

Escherichia coli O29:H21 is a human enterotoxigenic serotype that produces heat-stable (ST-I) enterotoxin, adheres diffusely to HeLa cells, and presents colonization factor antigen IV (CFA/IV) composed of CS5CS6 surface antigens. In one strain studied the genes for diffuse adherence and CFA/IV (CS5CS6) production were found to be present in the same plasmid encoding ST-I. The virulence plasmid (Ent) presented two unrelated basic replicons homologous to repFIC and repW. Gene(s) encoding diffuse adherence did not share homology with the probe for F1845 fimbrial adhesin which is responsible for this phenotype in other E. coli strains. Ent plasmids containing genes for diffuse adherence have not been described previously.     

Pharmacokinetics, oral bioavailability, and metabolism in mice and cynomolgus monkeys of (2'R,5'S-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, an agent active against human immunodeficiency virus and human hepatitis B virus

(2'R,5'S-)-cis-5-Fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine (524W91) is a nucleoside analog with potent anti-human immunodeficiency virus and anti-human hepatitis B virus activities in vitro. The pharmacokinetics and bioavailability of 524W91 after oral dosing were studied in mice dosed with 10, 100, and 600 mg of 524W91 per kg of body weight by the oral and intravenous routes. Cynomolgus monkeys were dosed with 10 and 80 mg of 524W91 per kg. In both species, the clearance of 524W91 was rapid, via the kidney, and was independent of dose. In monkeys, the total body clearance of 10 mg of 524W91 per kg was 0.7 +/- 0.1 liter/h/kg, and the volume of distribution at steady state was 0.8 +/- 0.02 liter/kg. The terminal elimination half-life was 1.0 +/- 0.2 h. The absolute bioavailability after oral dosing was 63% +/- 4% at 10 mg/kg. Concentrations of 524W91 in the cerebrospinal fluid were 4% +/- 0.7% of the corresponding levels in plasma. In mice, the total clearance of 10 mg of 524W91 per kg was 2.3 liters/kg/h, and the volume of distribution at steady state was 0.9 liter/kg. Absolute bioavailability in mice after oral dosing was 96% at a dose of 10 mg/kg. The metabolism of orally administered [6-3H]524W91 was studied in cynomolgus monkeys at a dose of 80 mg/kg and in mice at a dose of 120 mg/kg. Monkeys excreted 41% +/- 6% of the radioactive dose in the 0- to 72-h urine, 33% +/- 10% in the feces, and 10% +/- 7% in the cage wash. Unchanged 524W91 was 64% of the total radiolabeled drug recovered in the urine. The glucuronide was a minor urinary metabolite. 5-Fluorouracil was not detected (less than 0.02% of the dose). Mice dosed orally with 120 mg of [6-3H]524W91 per kg excreted 67% +/- 7% of the radiolable in the )- to 48-h urine. Small amounts of the 3' -sulfoxide and glucuronide metabolites were observed in the urine, but 5-fluorouracil was not detected. Good bioavailability after oral dosing and resistance to metabolism recommend 524W91 for further preclinical evaluation.     

Ryanodine action at calcium release channels. 1. importance of hydroxyl substituents

Ryanodine (1) and dehydroryanodine (2) have a polar face formed by cis-hydroxyls at C-2, C-4, C-6, and C-12. The importance of the hydroxyls to the action of 1 and 2 at the ryanodine receptor (ryr) of calcium release channels is examined at [3H]-1 binding sites in brain and skeletal muscle and in heart membranes relative to cardiac contractility, a pharmacologic response which appears to be mediated by the ryr. Five types of changes are considered: blocking the 4- and 6-hydroxyls as cyclic borates and boronates; blocking the 10- and 12-hydroxyls as cyclic phosphates, phosphonates, and phosphoramidates; methylation at nitrogen or hydroxyls at C-4 and C-10; dehydration of the C-2 hydroxyl; additional data for a 4,12-oxygen-bridged series. The first change has little effect on potency possibly due to the lability of the boron protective groups whereas the cyclic phosphorus compounds have reduced activity. Methylation reduces potency the least at nitrogen and the C-4 hydroxyl. Dehydration of 1 to 2-deoxy-2(13)-dehydro-1 allows the restoration of oxygen at C-2 by conversion to epoxides or a diol. One of the epoxides and 2-deoxy-2(13)-dehydro-2 retain 8-31% of ryanodine's potency in the ryr assays and 81% in the cardiac contractility system. In the 4,12-oxygen-bridged series, high potency at the receptor and cardiac muscle is retained in the 4-hydroxy ketal.     

Cytogenetic abnormalities in pediatric myelodysplastic syndrome: a report of three cases

Three consecutive cases of pediatric myelodysplastic syndrome (MDS) diagnosed over a three-year period in Queen Mary Hospital, Hong Kong, were described. Depending on the classification system used, they comprised two cases of chronic myelomonocytic leukemia (CMMoL) of which one can be reclassified as juvenile chronic myeloid leukemia (JCML) and one cases of refractory anemia with excess of blasts (RAEB) or an alternative diagnosis of atypical CML. Cytogenetic abnormalities were detected in all of them on examination of bone marrow cells. Of the two CMMoL, one had monosomy 21, whereas the other had hypodiploidy. The patient with RAEB had a complex karyotype of 46,X,del(X)(q24),t(1;7) (p22;q32),add(15)(q26)(8). The balanced translocation (1;7) seen in this patient was exceedingly rare and, to the best of our knowledge, was reported only twice in the literature. The karyotypic abnormalities that we saw in our patients were not well recognized in pediatric MDS. This report emphasizes the importance of cytogenetic study in children suspected of suffering from MDS, which remains a rare disorder of childhood, and a need to rationalize current classification schemes.     

Characterization of lineage restricted forms of a Xenopus CD45 homologue

The leukocyte common antigen, also known as CD45, is a structurally heterogenous molecule ranging in molecular weight from 180 to 220 kDa. CD45 belongs to a family of high molecular weight, cell surface glycoproteins expressed on all hematopoietic lineages with the exception of mature erythrocytes. In higher vertebrates, the highly conserved cytoplasmic domain of CD45 exhibits protein tyrosine phosphatase activity and has been implicated in lymphocyte activation through dephosphorylation of critical tyrosine residues on substrates associated with signal transduction pathways. The monoclonal antibody CL21 recognizes a high molecular weight determinant expressed on the surface of Xenopus leukocytes which was postulated to be a CD45 homologue. In order to determine if lymphocyte subpopulations expressed different molecular weight variants, splenic B cells were identified and isolated on the basis of surface IgM and the CL21 determinant expressed by these cells was compared to the determinant expressed by thymocytes. Immunoprecipitation revealed that IgM + B cells expressed a 220 kDa molecular weight variant whereas thymocytes and IgM-cells expressed a 180 kDa variant. Bone marrow myeloid cells, isolated on the basis of light scatter properties, expressed a determinant which ranged from 150 to 160 kDa. Dephosphorylation experiments utilizing p-nitrophenyl phosphate, 32P-labeled Raytide [tyr(P)], or Kemptide [ser(P)] as substrates demonstrated that immunoprecipitated CL21 antigen exhibited tyrosine specific phosphatase activity which was inhibited by sodium orthovanadate. Thus, data based on the presence of enzymatic activity and lineage restricted molecular weight variants support the hypothesis that the CL21 determinant is the amphibian homologue of mammalian CD45, and suggest that both structural and functional elements of CD45 have been conserved during vertebrate evolution.     

A re-investigation of questionable subclassifications of presynaptic alpha2-autoreceptors: rat vena cava, rat atria, human kidney and guinea-pig urethra

It has been suggested that at least the majority of mammalian presynaptic alpha2-autoreceptors belong to the genetic alpha2A/D-adrenoceptor subtype. The aim of the present study was to re-examine the alpha2-autoreceptors in tissues in which previous assignments conflicted with this alpha2A/D rule: in the rat vena cava and rat heart atria, where the autoreceptors were classified as alpha2B or similar to, but not identical with, alpha2D, and in the human kidney, where they were classified as alpha2C. Also re-examined were the autoreceptors in the guinea-pig urethra, where they were suggested to be alpha2A, in agreement with the rule, but in contrast to indications that the alpha2A/D-adrenoceptor of the guinea pig possesses alpha2D pharmacological properties. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically under autoinhibition-free or almost autoinhibition-free conditions. The Kd values of up to 14 antagonists (including the partial agonist oxymetazoline) against the release-inhibiting effect of the alpha2 agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. UK 14,304 reduced the evoked overflow of tritium with an EC50 between 6.3 and 13.2 nM. All antagonists (except prazosin in one case) shifted the concentration-inhibition curve of UK 14,304 to the right. Comparison of the Kd values thus obtained with Kd values at known alpha2 subtypes indicated that the autoreceptors in the rat vena cava, rat atria and the guinea-pig urethra were alpha2D and those in the human kidney alpha2A. For example, the pKd values of the antagonists in the rat vena cava, in rat atria and in the guinea-pig urethra were closely correlated with pKd values at the prototypic alpha2D radioligand binding sites in the bovine pineal gland (r = 0.96, P < 0.001; r = 0.92, P < 0.01; and r = 0.95; P < 0.001) and with the pKd values at the alpha2D-autoreceptors of guinea-pig atria (r = 0.99, P < 0.001; r = 0.95, P < 0.001; and r = 0.98, P < 0.001). The pKd values at the autoreceptors in rat vena cava, rat atria and guinea-pig urethra were not significantly or more loosely correlated with pKd values at alpha2A, alpha2B and alpha2C binding sites and alpha2A-autoreceptors. On the other hand, the pKd values of the antagonists in the human kidney were closely correlated with pKd values at the prototypic alpha2A radioligand binding sites in HT29 cells (r = 0.95; P < 0.001) and with pKd values at the alpha2A-autoreceptors of the pig brain cortex (r = 0.97; P < 0.001), but were not significantly or more loosely correlated with pKd values at alpha2B, alpha2C and alpha2D binding sites and alpha2D-autoreceptors. In contrast to previous suggestions, the autoreceptors in rat vena cava and atria are alpha2D, those in the human kidney alpha2A, and those in the guinea-pig urethra equally alpha2D. All, therefore, conform to the rule that alpha2-autoreceptors belong at least predominantly to the genetic alpha2A/D subtype of the alpha2-adrenoceptor. The apparent paradox of an alpha2A-autoreceptor in the urethra of the guinea pig, a species in which the genetic alpha2A/D-adrenoceptor otherwise has alpha2D pharmacological properties, is removed.     

Unindicated preoperative testing: ASA physical status and financial implications

Adenoid basal carcinoma of the uterine cervix is rare and its cell origin is still obscure. We report a case of adenoid basal carcinoma of the uterine cervix discovered incidentally in a 69-year-old woman who had been hysterectomized due to endometrial adenocarcinoma of the uterine corpus. Histologically, small round-to-oval cancer cell nests with peripheral cell palisading were seen budding from the basal cell layer of the uterine cervix showing carcinoma in situ. Immunohistochemically, the basaloid cells of the adenoid basal carcinoma were positive for keratins 14, 17 and 19 and resembled reserve cells of the cervical epithelium. The results of this study clearly demonstrated that adenoid basal carcinoma shows a phenotype similar to reserve cells of the uterine cervix. A review of the literature indicated that this tumor has a favorable prognosis and should be clearly separated from adenoid cystic carcinoma, which has a much poorer outcome.     

Overexpression of Rab3D enhances regulated amylase secretion from pancreatic acini of transgenic mice

Rab3D, a member of the ras-related GTP-binding protein Rab family, is localized to secretory granules of various exocrine tissues such as acinar cells of the pancreas, chief cells of the stomach, and parotid and lacrimal secretory cells. To elucidate the function of Rab3D in exocytosis, we have generated transgenic mice that over-express Rab3D specifically in pancreatic acinar cells. Hemagglutinin-tagged Rab3D was localized to zymogen granules by immunohistochemistry, and was shown to be present on zymogen granule membranes by Western blotting; both results are similar to previous studies of endogenous Rab3D. Secretion measurements in isolated acinar preparations showed that overexpression of Rab3D enhanced amylase release. Amylase secretion from intact acini of transgenic mice 5 min after 10 pM cholecystokinin octapeptide (CCK) stimulation was enhanced by 160% of control. In streptolysin-O-permeabilized acini of transgenic mice, amylase secretion induced by 100 microM GTP-gamma-S was enhanced by 150%, and 10 microM Ca2+-stimulated amylase secretion was augmented by 206% of that of the control. To further elucidate Rab3D involvement in stimulus-secretion coupling, we examined the effect of CCK on the rate of GTP binding to Rab3D. Stimulation of permeabilized acini with 10 pM CCK increased the incorporation of radiolabeled GTP into HA-tagged Rab3D. These results indicate that overexpression of Rab3D enhances secretagogue-stimulated amylase secretion through both calcium and GTP pathways. We conclude that Rab3D protein on zymogen granules plays a stimulatory role in regulated amylase secretion from pancreatic acini.