Regulation of canine gastric mucin synthesis and phospholipid secretion by acid secretagogues. Key components of the mucous gel include the glycoprotein mucin and surface-active phospholipids. In the present study, mucin production and release of the surface-active phospholipid phosphatidylcholine (PC) into the medium were measured with an isolated canine mucous cell culture system. Stimulation of glycoprotein synthesis in response to 10(-4) mol/L histamine (160% +/- 9% of control, P < 0.01), 10(-6) mol/L gastrin (129% +/- 7%, P < 0.01), and 10(-6) mol/L carbamylcholine (129% +/- 7%, P < 0.01) was observed by metabolic labeling, whereas prostaglandin E2 (PGE2) had no effect. The effect of histamine was blocked by the H2 receptor antagonist cimetidine but not the H1 receptor antagonist diphenhydramine (P < 0.01). Activators of adenylate cyclase and cyclic adenosine monophosphate analogs significantly stimulated mucin synthesis (P < 0.05). A 7.8% +/- 1.7% increase in mucin above basal levels after 24 hours was observed with a solid-phase immunoassay in control wells, whereas histamine, gastrin, and carbamylcholine increased total mucin by 14% +/- 0.7%, 17% +/- 4.3%, and 20.4% +/- 4%, respectively (all P < 0.01), and PGE2 had no significant effect. PC release was stimulated by the administration of histamine, carbamylcholine, gastrin (108%-110% of control, P < or = 0.05), and PGE2 (120% of control, P < 0.01). The acid secretagogues histamine, gastrin, and carbamylcholine stimulated mucin synthesis and PC release. PGE2 has no direct role in the synthesis of canine gastric mucin but stimulates release of surface-active phospholipids. The mechanisms responsible for acid secretion provide for the coordinated production of the primary layer of defense against the injurious effects of low pH.


A particular P-element insertion is correlated to the P-induced hybrid dysgenesis repression in Drosophila melanogaster. Transposable P elements in Drosophila melanogaster cause hybrid dysgenesis if their mobility is not repressed. The ability to regulate the dysgenic activity of the P elements depends on several mechanisms, one of which hypothesized that a particular deleted P element (the KP element) results in a non-susceptibility which is biparentally transmitted. In this study totally non-susceptible lines, and susceptible lines containing exclusively KP elements (IINS2 line and IIS2 line) were isolated from a M' strain. We show that non-susceptibility is correlated with a particular insertion of one KP element located at the cytological site 47D1. The repression ability of the GD sterility is determined by a recessive chromosomal factor, and cannot be due to the KP-element number. Here the repression of the P mobility is associated with reduction of the P transcripts and the inhibition of P promoter activity.


Distribution and structure of cloned P elements from the Drosophila melanogaster P strain pi 2. P transposable elements of Drosophila melanogaster cloned from the strong P strain pi 2 have been analysed. The structures and chromosomal locations of 26 of the 30-50 elements estimated to be present in pi 2 have been determined. At one location two elements are inserted 100 base pairs (bp) apart, and in a second location two elements are only separated by the 8 bp duplicated upon P-element insertion. In addition to 2.9 kilobase-pair (kbp) elements, elements with 14 different internal deletions from 1.3 to 2.3 kbp in size have been isolated. There are 7 copies of the 2.9 kbp element, 2 copies each of 5 internally deleted elements and a single copy of 9 internally deleted elements. One of the elements found twice is the KP element, which may play a role in the regulation of hybrid dysgenesis in strains which contain many copies of this element. Apart from internal deletions the elements are extremely homogeneous in DNA sequence, with only 2 single base polymorphisms detected twice each in over 16 kbp of P-element sequence. Although transpositions are infrequent in an inbred P cytotype strain such as pi 2, the distribution of these cloned elements indicates that when the genomic library was made, the strain was polymorphic with respect to element location. The distribution and structures of the element are discussed with respect to models for regulation of P-element transposition.


The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase. The gene URA6 encoding uridylate kinase (UK) from Saccharomyces cerevisiae was isolated as a multicopy suppressor of the respiratory-deficient phenotype of an S. cerevisiae mutant defective in the gene AKY2 encoding AMP kinase (AK). The URA6 gene also restored temperature resistance to two different temperature-sensitive mutations in the gene encoding Escherichia coli AK. By contrast, the gene encoding UK of Dictyostelium discoideum on a multicopy yeast shuttle plasmid, expressed under control of the constitutive yeast AKY2 promoter, failed to complement the deficiency in yeast, although such transformants expressed high UK activity. We show that yeast UK exerts significant AK activity which is responsible for the complementation and is absent in the analogous enzyme from D. discoideum. Since UK also significantly phosphorylates CMP (but not GMP), it must be considered an unspecific short-form nucleoside monophosphate kinase. Wild-type mitochondria lack UK activity, but import AKY2. Since multicopy transformation with URA6 heals the Pet- phenotype of AKY2 disruption mutants, the presence of AKY2 in the mitochondrial intermembrane space is not required to maintain respiratory competence. However, furnishing UK with the bipartite intermembrane space-targeting presequence of cytochrome c1 improves the growth rates of AKY2 mutants with nonfermentable substrates, suggesting that AK activity in mitochondria is helpful, though not essential for oxidative growth.


Molecular analysis of the yeast Ty4 element: homology with Ty1, copia, and plant retrotransposons. The element; Ty4 is a retrotransposon present in low copy number in the genome of Saccharomyces cerevisiae [Stucka et al., Nucleic Acids Res. 17 (1989) 4993-5001]. We have determined the complete nucleotide sequence of one such element from a particular strain and compared it to the other two elements occurring in this strain. The genomic organization of Ty4 is homologous to that found in other retrotransposons of the Ty1-copia group. The internal part of the element contains two large open reading frames (TY4A and TY4B) overlapping by 226 bp in a + 1 mode. TY4A reveals characteristics of the gag portion of retrotransposons and retroviruses, while TY4B consists of a protease, an integrase, a reverse transcriptase, and an RNase H domain (in that order). Our analyses suggest that only one of these copies might be transpositionally active. Sequence comparisons at the amino acid level show that the domains in Ty4 diverge considerably from those of other retrotransposons. The greatest similarity is seen between the reverse transcriptases (50%), the proteases (40%), and the integrases (30%) of Ty4, Ty1/2 and copia, respectively, whereas the degree of similarity for all other entities of these elements is much lower. Considering evolutionary aspects of the retrotransposons, we have to conclude that Ty4 has diverged at an early stage from the progenitors of other known retroelements and represents a novel and independent subgroup of the Ty1-copia class of retrotransposons.


Construction of a Tn7-lux system for gene expression studies in gram-negative bacteria. A Tn7-lux system was developed for gene expression studies in Gram- bacteria. The plasmids constructed, pHSK728 and pHSK729, have the following features: (1) a promoterless Vibrio fischeri lux operon as a reporter system; (2) multiple cloning sites (MCS) ahead of the lux operon, in opposite orientation for the cloning of promoter fragments; (3) a transcriptional terminator ahead of the MCS and translational stop codons in all reading frames before the translational start of the luxC gene; (4) a streptomycin/spectinomycin-resistance encoding gene as a selection marker; and (5) Tn7 border sequences flanking the above elements, permitting the transposition of lux fusion constructs into bacterial genomes. The system was tested using the Escherichia coli lac promoter as well as the differentially regulated promoters of the avrD gene from Pseudomonas syringae pv. tomato and the pelE gene of Erwinia chrysanthemi EC16. Southern blot analysis showed that all fusion constructs had integrated into the host genomes in a single-copy, site-specific manner. The promoters of the avrD and pelE genes resulted in little or no light production when bacteria were grown in rich culture media, but high levels of induction were observed when the bacteria were grown in plant tissues. These results demonstrated that the Tn7-lux system provided a simple, sensitive assay of promoter activity in Gram- bacteria.


Occurrence of dysplasia and adenocarcinoma after experimental chronic ulcerative colitis in hamsters induced by dextran sulphate sodium. In this study, long term dextran sulphate sodium administration was studied to ascertain whether colorectal carcinoma could be produced in patients with long standing ulcerative colitis. Simultaneously, changes in the intestinal microflora were analysed. Low grade to high grade dysplasia was seen in three of the five hamsters treated with 1% dextran sulphate sodium solution for 100 days, while no dysplasia was detected in the eight animals concomitantly treated with metronidazole, an antianerobic microbial agent, which prevents colonic ulceration. In these two groups, none of the animals developed colorectal cancer over 100 day period. In a group treated for 180 days, seven of the eight animals had dysplasia, and one had two adenomas. Furthermore, four of the eight animals had adenocarcinoma in the transverse colon; they were protruding well differentiated adenocarcinoma in one and non-protruding lesions infiltrating into the musclaris propria in three. The three non-protruding infiltrating adenocarcinomas were classified to be well differentiated adenocarcinoma in one and mucinous adenocarcinoma in two, resembling the type of cancer which complicates ulcerative colitis in man.


Inhibition by salbutamol of GHRH-induced GH release in type 1 diabetes mellitus. Patients with type 1 diabetes mellitus (IDDM) show augmented GH secretion, which is implicated in the pathogenesis of microvascular complications. On the other hand, it is well known that beta-adrenergic receptors have inhibitory influence on GH secretion, likely via stimulation of hypothalamic somatostatin. Since the possibility of pharmacological suppression of GH secretion would be of value in IDDM, we investigated the effect of salbutamol (SAL, 4 mg orally at -60 min) on the GH response to GHRH (1 micrograms/kg iv at 0 min) in 6 well-controlled (mean HbA1c +/- SEM: 7.3 +/- 0.5%) patients with IDDM. Salbutamol was able to inhibit basal GH levels (p < 0.05) as well as to abolish the GHRH-induced GH rise. After SAL administration, a significant (p < 0.05) reduction of glucagon levels was also found. Our data show that the enhancement of beta 2 adrenergic activity by oral therapeutical doses of SAL inhibits basal and GHRH-stimulated GH secretion in patients with IDDM.


Phenotypic properties of 3T3 cells transformed in vitro with polyoma virus and passaged once in syngeneic animals. Cloned BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) acquired a higher tumorigenicity phenotype after a single in vivo passage. Some of the in vivo passaged cells (CTC cells) exhibited also a higher metastatic phenotype than cells from the same clones that were maintained only in culture (C cells). A phenotypic comparison between CTC and C cells was performed. It was found that most CTC lines exhibited a higher binding to laminin compared to their clonal C cell ancestors. Some CTC cells were less sensitive to the cytotoxic effects of TNF-alpha than the corresponding C cells. CTC cells originating from tumors which appeared after a long latency period (late tumors) tended to express Fc gamma RII while CTC cells originating from tumors which appeared after a short latency period (early tumors) as well as the corresponding C cells tended not to express Fc gamma RII. The expression of a membrane epitope recognized by a monoclonal antibody expressing specificity towards PyV transformed cells, was down-regulated on late tumor cells compared to early tumor cells. Transfection of cloned PyV-transformed BALB/c 3T3 cells with the beta 1Fc gamma RII gene augmented the tumorigenicity and metastatic phenotype of the transfectants compared to control transfectants.


The diversity of retroviral diseases of the immune system. Retroviruses have been implicated as causative agents for a range of diseases including neoplasia, autoimmunity and immunosuppression. No two retroviruses carry the same complement of genes and for this reason it is not surprising that they induce a variety of different disease states. One common element in retroviral evolution has been the need to avoid immune recognition in order to persist within the host. A comparative approach, looking at various persistent retroviruses, has been used to pin-point the types of genetic adaptations adopted by retroviruses to remain hidden, often within the T cell compartment. Most of these retroviruses are T-cell-tropic and the diseases which they induce usually reflect the effect of the retrovirus on normal lymphocyte function.


Angiotensin and angiotensin converting enzyme tissue levels in two-kidney, one clip hypertensive rats. Renal tissue angiotensin I (Ang I) and II (Ang II) content and angiotensin converting enzyme activity were assessed in both kidneys during initial (7 days) and maintenance (25 days) phases of two-kidney, one clip hypertension in rats. At 7 and 25 days, systolic arterial pressure was 146 +/- 2 and 170 +/- 7 mm Hg, respectively. After 7 days, Ang I content of clipped kidneys was 64% and 70% higher (p < 0.001) than in nonclipped and sham-operated kidneys, respectively, when compared with levels in kidneys from sham-operated rats. In kidneys harvested 25 days after clipping one renal artery, Ang I and Ang II contents in clipped kidneys were increased 102% and 24% (p < 0.01), respectively. Ang II content was also 32% higher in nonclipped kidneys. Angiotensin converting enzyme activity in nonclipped kidneys was greater (p < 0.05) than that in either clipped (46% higher) or sham-operated kidneys (57% higher). Plasma Ang I and Ang II levels were elevated at 7 days but were not different at 25 days in clipped rats. These results demonstrate a dissociation between intrarenal and circulating levels of Ang I and Ang II and suggest that qualitatively different mechanisms may be responsible for the elevated intrarenal Ang II levels during the initial and maintenance phases of renal hypertension.


Regulation of renin gene expression in rat adrenal zona glomerulosa cells. Our previous studies indicated that the amount of renin present in cultured adrenal zona glomerulosa cells increased after stimulation with adrenocorticotropic hormone or potassium. In the present study, we investigated the effects of adrenocorticotropic hormone or potassium on renin gene expression in cultured rat adrenal zona glomerulosa cells. The amount of rat renin messenger RNA (mRNA) was measured by complementary DNA synthesis and the competitive polymerase chain reaction method. The effects of adrenocorticotropic hormone or potassium on adrenal zona glomerulosa cell renin activity and renin mRNA content were compared with the activity and content of control cells. After 1 and 4 hours of stimulation by adrenocorticotropic hormone or potassium, total renin in the medium increased slightly; at the same time, the percent change in the amount of renin mRNA was 281% and 291%, respectively, in the adrenocorticotropic hormone-stimulated group and 218% and 348%, respectively, in the potassium-stimulated group. Twenty-four hours after adrenocorticotropic hormone or potassium stimulation, total renin in the medium increased significantly, by 689% and 220%, respectively; percent change in the renin mRNA content was 754% and 278%, respectively. These results demonstrate that adrenocorticotropic hormone and potassium increased the activity of adrenal renin through an increase in the level of renin mRNA.


Angiomatoid malignant fibrous histiocytoma of the eyelid. A 20 year old female had an angiomatoid malignant fibrous histiocytoma of her left upper eyelid extending into the orbit, frontal and temporal regions. The tumor was excised and radiotherapy was given. Nine months follow-up did not reveal any recurrence.


Inhibition of Streptococcus mutans adherence to saliva-coated hydroxyapatite by human secretory immunoglobulin A (S-IgA) antibodies to cell surface protein antigen I/II: reversal by IgA1 protease cleavage. The effect of human secretory immunoglobulin A (S-IgA) and serum antibodies to surface protein antigen (Ag) I/II on the adherence of Ag I/II-bearing Streptococcus mutans and of free Ag I/II to saliva-coated hydroxyapatite (SHA) was investigated. The inhibition by S-IgA of binding of both S. mutans and free Ag I/II to SHA was dependent on antibody to Ag I/II. Essentially no difference was found between S-IgA1 and S-IgA2 with respect to antibody-dependent inhibition of Ag I/II binding to SHA, but S-IgA1 inhibited S. mutans adherence more effectively than did either serum immunoglobulin A1 (IgA1) or IgG antibodies. The antiadherence effect of S-IgA was abrogated after cleavage by IgA1 protease. Purified Fab alpha fragments containing Ag I/II-binding activity enhanced the binding of free Ag I/II to SHA and showed greater binding to SHA than did intact S-IgA1. Despite its relative inability to interact with precoated SHA, S-IgA1 containing antibody to Ag I/II was readily incorporated into the salivary pellicle during coating, but this did not promote Ag I/II binding. These data suggest that S-IgA antibodies can inhibit the initial adherence of S. mutans to salivary pellicle-coated tooth surfaces in an adhesin-specific fashion, but the presence in the oral cavity of bacterial IgA1 proteases would potentially interfere with this antiadherence mechanism.


Rickettsia rickettsii induces superoxide radical and superoxide dismutase in human endothelial cells. Human endothelial cells infected with Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever, undergo striking morphological changes to the endoplasmic reticulum-outer nuclear envelope complex. These changes are accompanied by concurrent accumulation of intracellular peroxides. Both of these findings are consistent with the notion that cells undergo some form of oxidative stress. Since oxidant injury is often initiated or mediated through oxygen radicals, we examined superoxide radical generation when endothelial cells were exposed to R. rickettsii. We also examined the levels of superoxide dismutase, an enzyme induced in response to increased superoxide formation. The levels of both superoxide and superoxide dismutase increased when endothelial cells were exposed to R. rickettsii. These results, together with our previous findings, support our hypothesis that cells infected by this intracellular bacterium experience oxidant-mediated injury that may eventually contribute to cell death.


Nutritional interactions between two suspected periodontopathogens, Treponema denticola and Porphyromonas gingivalis. A mutual symbiotic enhancement of growth of Porphyromonas gingivalis and Treponema denticola is described in this report. Brain heart infusion broth supplemented with vitamin K did not support the individual growth of P. gingivalis or T. denticola. However, when inoculated as a mixture, both bacterial species did grow significantly. The growth-stimulating factors produced by P. gingivalis and T. denticola were dialyzable and heat stable and were further identified as isobutyric acid and succinic acid, respectively. Since some forms of periodontal disease are associated with the presence, in affected sites, of high numbers of P. gingivalis and spirochetes, it is suggested that the bacterial interaction described in this report might be of utmost importance in the initiation and progression of the disease.


Prostanoids synthesis in lymphocyte subpopulations by adrenergic and cholinergic receptor stimulation. The release of eicosanoids (PGE2 and TXB2) as a consequence of specific neurotransmitter receptor agonist stimulation is described herein. The differential expression of beta adrenergic and muscarinic cholinergic receptors on sets and subsets of lymphocytes was first identified. Saturation assays with a specific radioligand for beta adrenergic receptors (3H-DHA) showed that B-, T-, T-helper (Th) and T-suppressor/cytotoxic (Ts/c) lymphocyte enriched populations all displayed beta adrenergic receptors. In contrast, when a specific radioligand for muscarinic cholinergic receptors (3H-QNB) was used, B-lymphocytes showed a lack of high-affinity muscarinic cholinergic receptors, while T-lymphocytes expressed them. Ts/c murine lymphocytes had more muscarinic cholinergic receptors than did Th cells. Specific receptor stimulation by the agonist caused a release of different eicosanoids depending on the cell type. Isoproterenol, triggered the release of TXB2 by B and Th-cells, but had no effect on Ts/c-cells. On the other hand, the muscarinic cholinergic agonist, carbachol only induced the release of PGE2 by Ts/c-cells. These results suggest differences in the expression and function of neurotransmitter receptors in sets and subsets of murine lymphocytes regarding the release of eicosanoids.


Ion chromatographic analysis of high specific activity 18FDG preparations and detection of the chemical impurity 2-deoxy-2-chloro-D-glucose. Because of the widespread use of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) prepared by the "Julich" method or its variants it was decided necessary to determine the major chemical impurities present in the final product. An analytical system for quantifying FDG was developed using pulsed amperometry after separation by high-performance anion exchange chromatography. With this system a heretofore unidentified impurity, 2-deoxy-2-chloro-D-glucose (C1DG, ca 20-2000 micrograms; typically < 100 micrograms), was found in our preparation and in those from other laboratories using the "Julich" method. C1DG arises from Cl- ion displacement during the labeling procedure where Cl- ion comes from several sources, and Cl- ion displacement from the HCl used in the hydrolysis step. FDG mass was present in the same preparations at a level of ca 1-40 micrograms. Other major chemical constituents were glucose (ca 1-6 mg) and mannose (ca 10-18 micrograms). Glycerol, arising from sterilizing filters, was also detected in most preparations. Although C1DG is a chemical impurity which has not been detected previously in nca FDG preparations, its biochemical and pharmacological properties are similar to FDG and 2-deoxy-D-glucose. Thus it is unlikely that the presence of small quantities of C1DG found in typical FDG preparations (ca 100 micrograms) would have adverse pharmacological or toxicological consequences that would limit continued application of this radiopharmaceutical in basic and clinical studies.


A simple synthesis of [11C]methyl triflate. [11C]Methyl triflate ([11C]methyl trifluoromethanesulfonate) was formed in high yield when [11C]methyl iodide in a nitrogen carrier was passed at 200 degrees C through a column containing graphitized carbon impregnated with 50% by weight of silver triflate.


Okadaic acid enhances human T cell activation and phosphorylation of an internal substrate induced by phorbol myristate acetate. Okadaic acid is a potent tumor promoter and an inhibitor of serine/threonine-specific protein phosphatases. We studied the effect of okadaic acid in human T cell activation and phosphorylation of internal substrates. Okadaic acid at up to 4 nM enhanced phorbol myristate acetate (PMA)-induced proliferation and CD25 (IL-2 receptor, p55) expression, although it showed no activation by itself. Okadaic acid induced hyperphosphorylation of a 60 kDa protein in T cells as well as non-T cells, as reported in fibroblasts and keratinocytes. Preincubation with 4 nM okadaic acid enhanced PMA induced phosphorylation of the 80 kDa protein, an internal substrate of protein kinase C in T cells. These results suggest that okadaic acid inhibited dephosphorylation of protein kinase C specific substrates, and as a result, enhanced T cell activation mediated by protein kinase C pathway.


The effect of TNFa on bradykinin receptor binding, phosphatidylinositol turnover and cell growth in human A431 epidermoid carcinoma cells. In this study, we examined the effect of TNFa on bradykinin (BK) B2 receptor binding and function in human A431 epidermoid carcinoma cells. [3H]BK binds to a single class of receptors on A431 cells in a saturable and reversible manner. A binding affinity (KD) of 3.0 +/- 0.3 nM (n = 4) and a Bmax of 151 +/- 14 fmols/10(6) cells, representing approximately 90,000 BK receptors per cell, was observed. The rank order of potency for BK agonist peptides indicates that the A431 BK receptor appears to be of the B2 subtype. When A431 cells were incubated with TNFa (10 ng/ml) for 48 h prior to BK binding, a significant decrease in the number of BK receptors compared to control was observed. TNFa did not influence the affinity of BK binding to A431 cells and direct addition of TNFa to the binding assay did not effect BK binding. BK-stimulated IP1 formation appeared to be increased in TNFa treated cells compared to control whereas histamine-stimulated IP1 formation was not influenced. Both control and TNFa treated cells were greater than 95% viable. However, TNFa treated cells were blocked in the G1 phase of the cell cycle resulting in a decrease in DNA synthesis. This may be one mechanism for the TNFa-induced decrease in BK receptors in A431 cells.


Development of 3-iodophenylisothiocyanate for radioiodination of monoclonal antibodies. A new radioiodination reagent, 3-iodophenylisothiocyanate (3-IPI) has been developed for coupling to monoclonal antibodies. The starting material, 3-tri-n-butylstannylphenylisothiocyanate was prepared via a reaction of hexabutylditin with 3-bromoaniline, followed by treatment with thiophosgene with an overall yield of 72%. The radioiodination of this tin precursor with Na[125I]I/iodogen in chloroform gave 3-[125I]IPI in 23-55% radiochemical yield and 81-99.6% radiochemical purity. Purification of the impure product by high pressure liquid chromatography increased the radiochemical purity of the product up to 99%. These results suggest that 3-IPI may be a useful ligand for radioiodination and coupling to a variety of monoclonal antibodies.


Targeting of [111In]biocytin to cultured ovarian adenocarcinoma cells using covalent monoclonal antibody-streptavidin conjugates. Three monoclonal antibodies (mAb) directed against the human ovarian adenocarcinoma cell line HEY, were substituted with maleimide and covalently bonded to thiolated streptavidin. The conjugates were separated from unreacted reagents by successive affinity chromatography on protein A-Sepharose and iminobiotin columns. Purified conjugates consisted of an immunoglobulin (Ig) monomer bound to a streptavidin tetramer through a covalent bond between the Ig molecule and one of the streptavidin subunits. The conjugates were able to specifically target [111In]biocytin to HEY cells in vitro in the presence of human serum and ascitic fluid from ovarian cancer patients.


Acetaminophen inhibits the human polymorphonuclear leukocyte function in vitro. The aim of the study is to investigate the effect of Acetaminophen (Am) on the oxidative respiratory burst of isolated human polymorphonuclear leukocytes (PMNs). Acetaminophen inhibited the luminolchemiluminescence (CL) peak response of PMNs stimulated with phorbol myristate acetate (PMA) or opsonized zymosan in a concentration dependent manner. The inhibitory effect of Am on PMA-stimulated PMNs-CL response was partially reversible. The level of CL inhibition with Am plus the hydroxyl radical scavengers allopurinol, dimethyl sulfoxide (DMSO) or superoxide dismutase (SOD) is greater than that with Am alone. Generation of superoxide (O2-) by stimulated PMNs, as assayed by superoxide dismutase inhibitable reduction of Ferricytochrome c, was markedly inhibited by Am. Furthermore, the phagocytic activity of PMNs as tested for by the ingestion of opsonized dead yeast was significantly reduced in Am-treated cells. These results indicate clearly that Am causes significant inhibition of the human PMNs function in vitro.


AO 1535 inhibits O2- production by human macrophages. AO 1535 is a semisynthetic monoglycosylceramide derived from O-glycosilated sphingosine, with a chemical structure similar to the glycolipids present in many mammalian tissues. In the epidermis monoglycosylceramides contribute to consolidate the structure of cutaneous layers. It has been recently shown that sphingosine and its derivatives are potent inhibitors of Protein kinase C, and block the 'respiratory burst' of phagocitic cells. In macrophages, like in neutrophils, the reactive oxygen intermediates are produced by a membrane associated enzymatic complex, NADPH-oxidase, which is activated by Protein kinase C. This study demonstrates that AO 1535 is able to inhibit the production of reactive oxygen intermediates in human monocytes and macrophages stimulated by phorbol ester and chemotactic tetrapeptide, suggesting a potential clinical application of AO 1535 in the treatment of inflammatory dermatoses.


Nuclear receptor sites for vitamin D-soltriol in midbrain and hindbrain of Siberian hamster (Phodopus sungorus) assessed by autoradiography. Autoradiograms were prepared from midbrains and hindbrains of male and female Siberian hamsters (Phodopus sungorus), kept under short-day or long-day illumination, after injection of tritium-labeled 1,25-dihydroxycholecalciferol (vitamin D, soltriol). Concentration and retention of radioactivity was noted in nuclei of certain neurons, glial cells, and ependymal cells, and in choroid epithelium. Labeled neurons of varying intensity were found throughout the brainstem in distinct populations at characteristic topographical sites, which include cranial nerve motor nuclei, the nucleus (n.) reticularis tegmenti pontis, the caudoventral region of the n. raphe dorsalis, the n. trapezoides, the n. vestibularis lateralis and n. vestibularis superior, neurons in the various nuclei of the sensory trigeminus, accessory optic nuclei, scattered neurons in nuclei of the reticular formation, the n. ambiguus, certain cells in the area postrema, and many others. Glial cells with nuclear labeling, probably microglia, were scattered predominantly in or near myelinated nerve fascicles. The choroid epithelium showed strong nuclear labeling throughout the ventricle. Nuclear labeling of ependyma was variable and weak, mainly at ventral and lateral extensions (recesses) of the ventricle. The extensive presence of nuclear binding in select neural structures indicates that vitamin D exerts specific genomic effects on cell populations that are known to be involved in the regulation of motor, sensory, autonomic, neuroendocrine, metabolic, and immune functions. The results of these studies, in conjunction with those from other brain and peripheral tissues, recognize vitamin D-soltriol as a steroid hormone with a wide scope of hormone-specific target cells, similar to estrogen, androgen, and adrenal steroids, and which are topographically distinct and characteristic for its functions as the steroid hormone of sunlight.


Quantitative immunogold localization of Na, K-ATPase along rat nephron. Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.


Detection of p53 gene mutations in human ovarian and endometrial cancers by polymerase chain reaction-single strand conformation polymorphism analysis. The presence of mutations in the p53 gene was examined in ovarian cancers by a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The primers were designed to amplify exons 5 through 9 that contain phylogenetically conserved domains of the p53 gene. Mutations were detected in 5 out of 10 cases, one of which contained a deletion in the second allele. A single base substitution was detected in 4 cases at codons 162, 175, 205 and 273 and a single base insertion in one case within codon 315. A high frequency of p53 mutations in ovarian cancers and lack of mutation in 6 benign ovarian tumors and 2 normal ovaries suggested that the mutation of the p53 gene was associated with the genesis and/or progression of ovarian cancer. In 1 of 7 endometrial cancers, two mutations at codons 239 and 254 were detected.


Coagulation abnormalities in liver disease. Hemostasis is intimately related to liver function, because most coagulation factors are synthesized by liver parenchymal cells and the liver's reticuloendothelial system serves an important role in the clearance of activation products. The extent of coagulation abnormalities depends upon the degree of disturbed liver function. Acute or chronic hepatocellular diseases may display decreases in the vitamin K-dependent factors (prothrombin; factors VII, IX, and X; proteins C and S), whereas other parameters remain normal. Patients with hepatic failure may present with the entire spectrum of factor deficiencies and may even develop disseminated intravascular coagulation (DIC). Patients with liver cirrhosis have a wide spectrum of abnormalities. Except for factor VIII:C and von Willebrand factor, all procoagulant and inhibitory factors are decreased, which is a reflection of impaired protein synthesis. Abnormal fibrinogen and prothrombin molecules can be identified. Platelets are quantitatively and qualitatively altered, and most patients develop DIC. Vitamin K deficiency leads to the production of abnormal vitamin K-dependent factors. The factors lack gamma-carboxy glutamic acid residues in the NH2-terminal part of their molecules. Surgery associated with the liver leads to major hemostasis alterations. The LeVeen shunt is invariably related to DIC. Bleeding with partial liver resection is mostly mechanically induced, but chronic DIC may be present. Orthotoptic liver transplantation is associated with severe hemorrhages. These are partly due to the pre-existing hemostasis defects and partly due to DIC with a marked fibrinolytic response. This is especially noted during the anhepatic phase and when the donor liver is perfused by the recipient's blood. Postoperative recovery is quick, provided the graft is not rejected. Postoperatively, there may be an initial hypercoagulable state, which could be related to the thrombosis occasionally encountered.


Multiple neoplasia in a patient with pituitary acromegaly. A case of pituitary acromegaly with multiple nonendocrine neoplasia is reported. The tumorogenic potential of growth hormone, prolactin and other growth factors is discussed.


The Lambert-Eaton syndrome.

The Lambert-Eaton syndrome. This rare myasthenic disorder is the first neurologic disease found to be caused by antibodies against an ion channel complex. It presents an unusual opportunity to clarify the pathobiology of so-called receptor diseases, as well as to gain better understanding of the normal biology of acetylcholine release and its effect on neuromuscular junctions.


High-performance liquid chromatography with electrochemical detection for the simultaneous determination of vitamin A, D3 and E in milk. A high-performance liquid chromatographic electrochemical detection for the rapid and simultaneous determination of the vitamin A, D3 and E is described. The separation is carried out by using a C18 reversed-phase column and 0.1 M LiClO4 in methanol-water (99:1, v/v) as the mobile phase. The compounds are eluted with good resolution in the above order within about 15 min and are determined by amperometric detection with a glassy carbon electrode at +1050 mV (vs. Ag/AgCl). The method gave reproducible results and the detection limits were of the order of 0.07, 4 and 0.2 ng of vitamin A, D3 and E, respectively. The method was successfully applied to the determination of vitamin A, D3 and E in liquid cow milk and milk powder samples. After saponification, fat-soluble vitamins were extracted with hexane and a methanolic solution of the dried extract was injected directly into the chromatographic system, avoiding the clean-up step that is necessary for vitamin D3 when electrochemical detection is not used. Good recoveries were obtained.


Demonstration of the distribution of coxsackie virus RNA in neonatal mice by non-isotopic in situ hybridization. A simple method for the demonstration of Coxsackie virus RNA by in situ hybridization is described. Oligonucleotides complimentary to conserved sequences of Coxsackie B genome were synthesised and labelled with digoxigenin using commercially available reagents. In addition to detecting all five Coxsackie B strains examined, six strains of Coxsackie A were also demonstrated by these probes. Using one of the oligonucleotides separately it was possible to distinguish Coxsackie A strains from the strains of Coxsackie B virus examined. This study demonstrates the presence of viral RNA in mice tissues showing morphological evidence of damage, confirming the suspected tropisms of these viruses. The method described is directly applicable to the study of the presence of these viruses in human tissue from diseases where a viral aetiology is suspected.


Monoclonal anti-idiotypes for the rapid detection of human cytomegalovirus. A novel enzyme-linked immunosorbent assay (ELISA) was developed for human cytomegalovirus (HCMV) utilizing a monoclonal anti-idiotype specific for CMVB1, an antibody to HCMV. Samples of HCMV were measured by their inhibition of the binding of CMVB1 to anti-idiotype. The ELISA detected HCMV in a concentration-dependent manner from 20 to 0.6 x 10(3) PFU/ml, with 50% inhibition at approx. 3 x 10(3) PFU/ml. These data demonstrate the potential of anti-idiotype antibodies as the basis of simple and rapid diagnostic tests for infectious agents.


Plasmid-associated effects on test gene expression and Marek's disease virus plaque formation during recombination trials. Marek's disease virus (MDV), a herpesvirus of avian origin, is being examined for suitability as a vector for expressing foreign genes. We observed that plasmids encoding the LacZ gene of E. coli under the control of either the herpes simplex virus alpha 4 immediate-early promoter or the cytomegalovirus major immediate-early promoter inhibited MDV plaque formation. Plaque numbers were decreased by one-third, and transient expression of the beta-galactosidase reporter gene was increased by up to 6-fold, when the plasmids were linearized. Sequences associated with the heterologous promoter were identified as being responsible for inhibiting MDV replication.


Detection of cytomegalovirus by a rapid culture system: a comparison of monoclonal antibodies in a clinical setting. A monoclonal antibody cocktail, directed against immediate early antigens of cytomegalovirus, was assessed in a rapid culture system for detection of CMV (DEAFF) in clinical samples. The antibody cocktail was shown to be equivalent in sensitivity and specificity to our currently used monoclonal antibody. A comparison of DEAFF (using either monoclonal preparation) and conventional cell culture shows that DEAFF detects a smaller proportion of CMV positive samples from those receiving antiviral chemotherapy. The reasons for the discrepancies observed between these techniques are discussed.


Detection of enhanced neutrophil adhesion to parainfluenza-infected airway epithelial cells using a modified myeloperoxidase assay in a microtiter format. Despite growing evidence that respiratory virus infections precipitate episodes of airway obstruction and airway hyper-responsiveness in young children and in asthma, little information is available on the mechanisms by which virus infections alter the airway physiology. Airway inflammatory changes (including influx of inflammatory cells such as neutrophils) have been described during episodes of airway hyper-responsiveness in both animal models and human subjects. Neutrophil damage to several cell types has been shown to require adhesion as a primary step. In order to examine the potential interactions between virus-infected airway epithelial cells and neutrophils, we have studied the ability of neutrophils to adhere to virus-infected airway epithelial cell cultures. Neutrophil adherence was determined indirectly, using myeloperoxidase as a marker for adherent neutrophils in an assay system described here. Airway epithelial cell cultures (both primary human tracheal epithelial cells, and two permanent cell lines, A549 and BEAS-2B) were grown in 96-well tissue culture plates and infected with human parainfluenza virus type 2. Infected airway epithelial cell cultures supported significantly enhanced levels of neutrophil adherence (up to 50-75% of neutrophils added to the wells) compared to uninfected control cultures. Moreover, this adherence occurred in a virus dose-dependent fashion, with increasing levels of adherence noted at increasing viral multiplicities of infection. The assay system described allows the detection of small numbers of adherent neutrophils (as few as 1000 neutrophils) in a 96-well format.


Molecular typing of ampicillin-resistant, non-beta-lactamase-producing Enterococcus faecium isolates from diverse geographic areas. Molecular typing methods were compared by using 66 ampicillin-resistant, non-beta-lactamase-producing Enterococcus faecium clinical isolates from diverse geographic areas. Whole-plasmid analysis, restriction enzyme analysis of plasmid DNA with EcoRI and HindIII, and contour-clamped homogeneous electric field electrophoresis with digestion by SmaI and ApaI were performed on all isolates. Whole-plasmid analysis identified 47 different groups. Restriction enzyme analysis of plasmid DNA identified 50 groups when EcoRI was used and 51 groups when HindIII was used. Results with EcoRI and HindIII differed in 9 of 66 isolates. Grouping results with whole-plasmid analysis differed from results of restriction enzyme analysis of plasmid DNA (combining EcoRI and HindIII) in 20 of 66 isolates. Contour-clamped homogeneous electric field electrophoresis identified 46 groups when SmaI was used and 44 groups when ApaI was used. Results with SmaI and ApaI differed in 3 of 66 isolates. Grouping results with contour-clamped homogeneous electric field electrophoresis (combining SmaI and ApaI) differed from results of restriction enzyme analysis of plasmid DNA (combining EcoRI and HindIII) in 17 of 66 isolates. The combined use of whole-plasmid analysis, restriction enzyme analysis of plasmid DNA with two enzymes, and contour-clamped homogeneous electric field electrophoresis with two restriction enzymes should be considered when E. faecium is typed for epidemiologic investigation.


Quantification of cytomegalovirus in bronchoalveolar lavage fluid after allogeneic marrow transplantation by centrifugation culture. A technique to quantify cytomegalovirus (CMV) by centrifugation culture of bronchoalveolar lavage fluid from marrow transplant recipients was developed. This technique was used to assess the CMV response to antiviral treatment and the relationship between viral load, asymptomatic excretion versus symptomatic infection, and prognosis. Relative to tube cell culture, centrifugation culture of bronchoalveolar lavage fluid was more sensitive than direct fluorescent-antibody staining. It was also a rapid, replicable method for detecting and measuring the amount of CMV. There was no significant difference between viral load at diagnosis and after 9 days of treatment with ganciclovir and intravenous immunoglobulin. Viral load was not predictive of outcome, and there was no difference in amount of virus between patients with asymptomatic CMV excretion and those with CMV pneumonia. The amount of CMV may not be as important as other factors (e.g., host immune response) in the pathogenesis of CMV pneumonia.


Cytomegalovirus (CMV) antigenemia assay is more sensitive than shell vial cultures for rapid detection of CMV in polymorphonuclear blood leukocytes. We compared the cytomegalovirus (CMV) antigenemia assay with shell vial cultures of polymorphonuclear leukocyte (PMNL)-enriched blood fractions for rapid diagnosis of CMV viremia. PMNL fractions of 280 blood specimens from 171 patients (170 solid-organ transplant recipients and 1 patient undergoing pretransplant evaluation) were inoculated in shell vial and conventional CMV cultures. A commercially available kit (CMV-vue kit; INCSTAR Corp.) was used for the CMV antigenemia assay, in which PMNL preparations were stained with monoclonal antibodies directed against the CMV protein pp65. Mixed-leukocyte blood fractions from the same blood specimens were inoculated in parallel shell vial and conventional cultures. CMV viremia (defined by the isolation of CMV in conventional cultures) was detected in 32 (13%) of 245 PMNL fractions included in the final analysis. Twenty-eight (87.5%) were also positive in the CMV antigenemia assay, whereas 22 (69%) were positive in shell vial cultures. Ten (4%) additional PMNL fractions positive only in the CMV antigenemia assay were from eight patients with active CMV infections (six patients), who had previous or subsequent episodes of CMV viremia (seven patients), or in whom CMV was isolated in cultures of simultaneously obtained mixed-leukocyte fractions (three patients). Overall, the CMV antigenemia assay was significantly more sensitive than shell vial cultures for detection of CMV in the PMNL fraction of blood leukocytes (P < 0.01, McNemar's test), and we recommend it as the method of choice for rapid diagnosis of CMV viremia.


Detection and quantification of latently infected B lymphocytes in Epstein-Barr virus-seropositive, healthy individuals by polymerase chain reaction. We designed a highly sensitive and specific polymerase chain reaction assay for the detection of Epstein-Barr virus (EBV)-related sequences in B-cell DNA of EBV-seropositive healthy individuals. By using this assay, we were able to amplify at least 10 copies of a plasmid containing the BamHI-W region, which is repeated up to 11 times within the EBV genome, in the presence of 1 microgram of EBV-negative DNA, indicating that one virus genome was detectable from 150,000 cells. In 15 of 16 tested individuals, EBV-related sequences were found frequently when the DNA from 10(6) B lymphocytes was examined and 1 microgram of DNA was used in each polymerase chain reaction. When the results of amplifying the diluted plasmid were used as a semiquantitative standard, the number of EBV genomes detected could be estimated to range between 50 and less than 1 from 10(6) B lymphocytes. The results of our study will provide the basis for further investigations of the characteristics of the latent carrier state in healthy EBV-seropositive individuals, such as the determination of the number of virus copies per cell and expression of antigens.


Analysis of ovine lentivirus infectivity and replication by using a focal immunoassay and an antigen-capture enzyme-linked immunosorbent assay. A focal immunoassay and an antigen-capture enzyme-linked immunosorbent assay (antigen-capture ELISA) were developed to quantify infectious ovine lentivirus (OvLV) and OvLV capsid protein (CA) (p27), respectively. The in vitro kinetics of replication and cytopathogenicity of distinct biological clones of OvLV (rapid/high and slow/low phenotypic variants) were assessed. Both viruses were detected by focal immunoassay within 48 h postinfection, 2 days before syncytia were observed in goat synovial membrane cells infected with rapid/high OvLV and 4 days before they appeared in cultures infected with slow/low OvLV. CA was first detected by antigen-capture ELISA in supernatants of cells infected with rapid/high OvLV 4 days postinfection, and it reached a plateau by 10 days, 4 days after peak syncytium formation. In contrast, in cultures infected at the same multiplicity of infection with slow/low OvLV, CA was detected 8 days postinfection, and the titer gradually increased over the following 12 days while the number of syncytia gradually decreased. Peripheral blood mononuclear cells (PBMC) from seropositive sheep treated with phorbol 12-myristate 13-acetate (PMA) generally expressed CA earlier and at higher levels than PBMC treated with either phytohemagglutinin or concanavalin A. Serum CA levels above 3 ng/ml were found in 58% (18 of 31) of seropositive sheep. However, there was no correlation between PMA-induced CA expression and levels of antigenemia. Viral heterogeneity may account for variations both in CA expression in cultures of PBMC and in antigenemia, humoral immune response, and viral pathogenicity in infected animals.


Rapid plasmid DNA isolation from mucoid gram-negative bacteria. Exopolysaccharides interfere with the isolation and characterization of plasmid DNA from gram-negative bacteria. To repress capsular polysaccharide production, bacteria were cultured in medium containing bismuth nitrate and sodium salicylate. Rapid removal of other contaminating bacterial surface components was achieved by mild acidic zwitterionic detergent extraction. After treatment, bacterial cells were more readily lysed in alkaline detergents. The resulting plasmid preparations contained virtually no capsular polysaccharide and relatively small quantities of lipopolysaccharide and protein, yet they produced yields of nucleic acids similar to those of conventional plasmid preparations. Conventional preparations from encapsulated organisms were largely insoluble and appeared as smears following agarose gel electrophoresis, with indefinite plasmid banding. Plasmids prepared by the new method were highly soluble in conventional buffers and exhibited high-resolution plasmid banding patterns in agarose gels. Plasmids as large as 180 kbp could be isolated and visualized, without apparent nicking, and were readily digested by restriction endonuclease enzymes. The method proved effective with encapsulated or mucoid strains of Klebsiella pneumoniae, Escherichia coli, Acinetobacter anitratus, Salmonella typhimurium, and Enterobacter species. The complete method for plasmid isolation was not suitable for Pseudomonas aeruginosa because of the inhibitory effects of bismuth. Thus, removal of contaminating bacterial surface structures enabled the rapid isolation and characterization of plasmids from mucoid clinical isolates, without the use of organic solvents, CsCl gradients, or expensive, disposable columns.


Comparison of the Vitek Immunodiagnostic Assay System with three immunoassay systems for detection of cytomegalovirus-specific immunoglobulin G. The Vitek Immunodiagnostic Assay System (VIDAS; bioMerieux Vitek Inc., Hazelwood, Mo.) was evaluated for its ability to detect anticytomegalovirus immunoglobulin G (IgG) and was compared with the following assay systems: Abbott IMx Cytomegalovirus IgG antibody assay (Abbott Laboratories, Abbott Park, Ill.), Whittaker Cytomegelisa II IgG assay (Whittaker Bioproducts, Walkersville, Md.), and Whittaker FIAX Cytomegalovirus IgG assay (Whittaker Bioproducts). Samples were considered positive if at least two of three (IMx, Cytomegelisa II, and FIAX) comparative assays gave positive results; a sample was considered negative if at least two of the three assays were negative. Of the 199 clinical serum samples tested, 194 gave concordant results among the three comparative assays (145 were positive and 49 were negative). The VIDAS results with two samples were persistently equivocal and were therefore excluded from the comparison study. The overall agreement between VIDAS and the established standard was 98.5%. The VIDAS assay yielded one false-positive and two false-negative results. The intra- and interassay precision studies indicate that the VIDAS assay is suitable for clinical use. The VIDAS system is rapid, self-contained, and fully automated and would be useful for screening of cytomegalovirus immune status.


Rapid determination of human cytomegalovirus susceptibility to ganciclovir directly from clinical specimen primocultures. We were able to rapidly determine the susceptibility of human cytomegalovirus to ganciclovir using a late antigen synthesis reduction assay directly on primocultures of clinical specimens. This test was compared with a conventional susceptibility assay which was performed with cell-free virus obtained after cytomegalovirus isolation and in vitro passages. Both tests produced similar results. The rapid test, unlike conventional assays, is able to provide a result within 5 days after receipt of the specimen and could thus play a direct role in the therapeutic decision.


Comparison of ViraPap, Southern hybridization, and polymerase chain reaction methods for human papillomavirus identification in an epidemiological investigation of cervical cancer. In order to provide a reliable diagnosis for the presence and type of human papillomavirus (HPV) DNA in a case-control study of cervical cancer in Colombia and Spain, 926 cervical scrapes from female subjects were examined by ViraPap (VP) and Southern hybridization (SH), and 510 of these (263 cases and 247 controls) were also tested by polymerase chain reaction (PCR) using the HPV L1 consensus primers. HPV DNA prevalence was much higher in cases than in controls by each of the three tests. There was complete agreement between the results of the three tests for 64.9% of the 510 specimens; 53.5% were negative and 11.4% were positive (regardless of type) by all tests. An additional 29.0% of the specimens were positive by PCR: 19.4% by PCR alone, 6.7% by PCR and VP, and 2.9% by PCR and SH. SH and/or VP gave positive results for 6.0% of the specimens for which the PCR finding was negative: 2.7% by SH alone, 2.5% by VP alone, and 0.8% by both VP and SH. When specimens which were positive by VP alone or only by SH at low-stringency conditions were excluded, PCR confirmed all but four specimens which were positive by other tests. The concordance between type-specific diagnosis by SH and PCR was 86% when HPVs were typed in both tests. HPV-16 accounted for over 80% of the typed HPVs in each test. The presence of blood in case specimens did not appear to inhibit HPV positivity by VP or by PCR at the dilution tested. Low amounts of cellular DNA of specimens resulted in some underestimation of HPV positivity by VP and SH but not by PCR. Compared with that of PCR, the sensitivities for case specimens were 38% by SH and 50% by VP; the sensitivity for control specimens, although it could not be measured precisely because there were few positive specimens, appeared to be lower than for case specimens. It was concluded that PCR-based tests are best suited for epidemiological investigation of HPVs.


Evidence of serologic diversity within group C rotaviruses. The Cowden strain of porcine group C rotavirus and the Shintoku strain of bovine group C rotavirus were classified as different serotypes by two-way cross-neutralization tests. Two neutralization patterns against the Cowden and Shintoku strains were observed when hyperimmune or convalescent-phase antisera to three noncultivatable porcine group C rotaviruses and a human group C rotavirus were used in one-way cross-neutralization tests. Antisera to two porcine group C rotaviruses and the human group C rotavirus neutralized the Cowden strain at high titers but did not neutralize the Shintoku strain, suggesting that these three strains are serotypically related to the Cowden strain. The remaining antisera to a porcine group C rotavirus (HF strain) reacted with the Cowden and Shintoku group C rotaviruses in cell culture immunofluorescence tests but did not neutralize either virus in one-way cross-neutralization, suggesting that the HF strain belongs to a third serotype. However, confirmation of these findings requires additional analysis by two-way cross-neutralization. Our findings support the existence of at least two distinct serotypes of group C rotaviruses, and possibly a third, among animals and humans. The serotypic similarity observed between the Cowden strain and a human group C rotavirus suggests that the cultivatable Cowden strain and antiserum to this virus may provide important reagents for the diagnosis of group C rotaviruses in humans.


Interpretive criteria and quality control parameters for testing susceptibility of Haemophilus influenzae to enoxacin, ofloxacin, and temafloxacin. Haemophilus influenzae isolates were uniformly susceptible to enoxacin, ofloxacin, and temafloxacin. Zone diameter and MIC interpretive criteria were proposed to define susceptible populations so that mutants with diminished susceptibility might be detected when and if they appear in clinical specimens. Additional collaborative quality control studies defined MIC and zone size limits for tests with H. influenzae ATCC 49247.


Coamplified positive control detects inhibition of polymerase chain reactions. Polymerase chain reactions (PCR) may fail to react because the substrate DNA is absent (true negative) or because of inhibition of specific amplification (false negative). The use of positive controls can increase confidence in negative PCR results by ruling out failure due to inhibition as a cause of the lack of amplification products. This report describes the construction and application of coamplified positive controls for herpes simplex virus and human herpesvirus 6 amplifications. Herpes simplex virus and human herpesvirus 6 PCR products were modified to generate control PCR products in which the original probe sequences were replaced by a Drosophila probe sequence. Implementation of the coamplified controls increased our specimen throughput in comparison with the parallel control amplifications used previously. Clinical laboratories using PCR for diagnosis of infectious diseases may find positive controls particularly helpful for increasing confidence that negative amplifications represent truly negative specimens.


Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus. We have noticed that suboptimal specimen processing and storage conditions may cause false-negative results in the detection of hepatitis C virus (HCV) RNA in plasma or serum. To establish the influence of specimen handling in a serological laboratory on the rate of detection of HCV RNA by the cDNA polymerase chain reaction (cDNA-PCR), we tested routine serum samples and fresh-frozen plasma samples from the same bleeding from confirmed anti-HCV-positive blood donors. When primers from the NS3/NS4 region were used, HCV RNA was detected in fresh-frozen plasma from 67% of the donors, whereas positive results were obtained with only 50% of the serum samples that had been subjected to routine serological procedures. Analysis of the same samples with primers from the highly conserved 5'-terminal region (5'-TR) revealed an HCV RNA detection rate of 92% for both the routine and the fresh-frozen samples. However, the yield of the amplification product in routine samples was strongly reduced compared with that in fresh-frozen plasma. Comparison of both primer sets for cDNA-PCR showed that the 5'-TR primer set was 10- to 100-fold more effective in detecting HCV RNA. We also analyzed the effect of storage of whole EDTA-blood and serum at room temperature and at 4 degrees C on the yield of the amplification product. A rapid decline in detectable HCV RNA of 3 to 4 log units was observed within 14 days when whole blood and serum were stored at room temperature. By contrast, no perceptible reduction in the cDNA-PCR signal was found in freshly prepared serum stored at 4 degrees C.


Rotavirus diarrhea in Bangladeshi children: correlation of disease severity with serotypes. To improve the understanding of the relative importance of serotypes of rotavirus in dehydrating diarrhea, we examined the correlation of clinical characteristics and disease severity with serotype in 2,441 diarrheal episodes among children younger than 2 years of age in rural Bangladesh. Of 764 rotavirus-associated episodes, a single G type (serotype 1, 2, 3, or 4) was determined by oligonucleotide probe in 485 (63%), while 233 episodes were nontypeable. Episodes with G types 2 and 3 were associated with more-severe dehydration than episodes associated with G type 1 or 4 or with nontypeable rotavirus. Episodes did not differ by G type in prevalence of vomiting, copious diarrhea, fever, abdominal pain, or length of treatment center stay. Rotavirus reinfections were detected in seven children, with homologous reinfection (G type 2) in one. Twelve children with diarrhea who died had rotavirus detected in stool specimens within 30 days of death. Children who died were more likely to be malnourished than were surviving children with rotavirus diarrhea. Of 40 specimens tested by polymerase chain reaction, 29 (72.5%) were P type 1, 9 (22.5%) were P type 2, 1 (2.5%) was P type 3, and 1 (2.5%) was nontypeable. One severely symptomatic diarrheal episode was associated with P type 3 rotavirus, a serotype usually found in asymptomatic nursery infections. Although G types 2 and 3 were associated with more-severe dehydration than other serotypes, the differences do not appear to be of major clinical importance. Effective vaccines should protect against all four major G types.


Nosocomial transmission of rotavirus from patients admitted with diarrhea. We studied the transmission of rotavirus (RV) in 950 patients under 2 years of age hospitalized for diarrhea in Santiago, Chile. Stool samples were collected every other day from all patients during their entire hospital stay. To trace nosocomial transmission, we mapped the ward at the time of detection of RV. Comparative study by polyacrylamide gel electrophoresis of 315 RV isolates (180 detected upon admission of patients and 135 attributed to nosocomial transmission) allowed the identification of 18 different electropherotypes. An electropherotype similar to that of a community-acquired case was found in the same room in 81% of nosocomial cases and in the ward in 92% of nosocomial cases. It was concluded that the infants admitted shedding RV are the major source of nosocomial transmission and there was not a RV strain that was particularly transmissible.


Methadone maintenance detoxification fear: a study of its components. These studies added three hypothesized fear components to the Detoxification Fear Survey Schedule (DFSS-14) and compared its psychometric properties to the original. Two disparate methadone maintenance populations (N = 226) were used in scale development. Thirty-one items and three factors emerged. A validation sample (N = 159) from two diverse methadone maintenance populations yielded a 27-item scale that discriminated between interview-diagnosed detoxification fear and non-fear (91.8% and 85.4% correctly classified). Finally, in methadone maintenance patients from three disparate programs in which the prevalence of detoxification fear was known, the DFSS-27 and DFSS-14 were compared. The DFSS-27 showed substantially improved sensitivity. The DFSS-27 seems a useful screen for detoxification fear in methadone maintenance, may aid intervention planning, and may prove a useful interventions outcome measure.


Severe thrombocytopenia secondary to asymptomatic cytomegalovirus infection in an immunocompetent host. A healthy 33 year old man presented with a short history of purpura and easy bruising. Investigations showed profound thrombocytopenia with atypical lymphocytes in the peripheral blood. Marrow appearances were consistent with platelet consumption. Biochemical hepatitis was also noted. An infection screen showed the underlying diagnosis to be cytomegalovirus (CMV) infection. He was treated successfully with oral prednisolone. This subsequently tailed off without relapse. Careful examination of a stained blood film is needed in all cases of apparent idiopathic immune thrombocytopenic purpura.


The occurrence of fungi in bovine tissues in relation to portals of entry and environmental factors. The occurrence of fungi in tissue specimens from 72 cattle was examined by culture, histopathology and indirect immunofluorescence staining (IIF). Groups of 12 animals each had been fed either concentrate or roughage and had been housed either in tie stalls, on slatted floors or on deep bedding. Specimens were obtained from the lung, omasum and Peyer's patches of the ileum and corresponding lymph nodes. Both hyphae and spores were made visible by IIF and by combination of IIF, morphology and conventional staining it was possible to differentiate between aspergilli and zygomycetes. In the lungs, aspergilli were detected at the same rate by morphology and IIF, whereas zygomycetes were found nearly twice as often by IIF than by culture. Fungi in pulmonary tissue were most frequent in cattle tied or kept on deep bedding (P < 0.01) as assessed by IIF. Within lymph nodes only spores were found, and Aspergillus fumigatus was the predominant species. Lesions devoid of fungi, especially ulcerations, were observed on the edges of the largest omasal laminae with a notable preference for the aboral third. The localization and histopathology suggested a reflux of acid abomasal contents to be the pathogenic principle. Granulomas with centrally located plant material were found more frequently in cattle fed roughage than in cattle fed concentrate (P = 0.01) and were differentiated from mycotic granulomas.


Specific diagnosis of parvovirus enteritis in dogs and cats by in situ hybridization. In a retrospective study, the fixed intestines of 10 dogs and 10 cats with intestinal lesions characteristic of parvovirus infection were assayed for the presence of parvovirus by in situ hybridization and immunohistochemistry. Parvoviral nucleic acid was localized by in situ hybridization in intestinal tissue in all 10 dogs and in nine of the 10 cats, whereas antigen was detected only in seven of 10 canine and eight of 10 feline intestines by immunohistochemistry. We conclude that an aetiological diagnosis can be established with a high degree of certainty by routine histology. Demonstration of the infectious agent by in situ hybridization, however, proves to be a valuable specific tool which allows an exact cellular localization of parvovirus in formalin-fixed, paraffin wax-embedded tissue sections.


Influence of dietary fiber and buffer value index on the ruminal milieu of lactating dairy cows. The influence of dietary buffer value index and dietary ADF content on ruminal fluid pH, buffering capacity, and buffer value index was measured. Four lactating Holstein cows (two primiparous) averaging 72 +/- 60 DIM were used in a 4 x 4 Latin square with 3-wk experimental periods. Treatments were a 2 x 2 factorial arrangement of TMR containing two ADF concentrations (16 and 21% of DM) and two buffer value indexes (calculated from analysis of individual dietary ingredients to be -200 and 0). Milk fat content and milk fat yield tended to be increased by high ADF, and protein yield tended to increase with low buffer value index and low ADF. Although the high ADF diets increased ruminal fluid pH, they reduced buffering capacity; because the magnitude of the pH increase was greater than the reduction in buffering capacity, ruminal fluid buffer value index was increased by added ADF. The high buffer value index diets reduced ruminal fluid pH and increased ruminal fluid buffering capacity; effects on pH outweighed those on buffering capacity so that the ruminal fluid index paradoxically decreased as the dietary index increased. Ruminal fluid acetate increased and propionate decreased as ADF increased. We conclude that ruminal fluid buffer value index increases with dietary ADF, likely because of reduced ruminal concentrations of fermentation acids. Because diets with the highest index produced the lowest ruminal indexes, dietary buffer value index must be studied further before it can be included in any model purporting to predict the need for supplemental dietary buffers.


Sodium bicarbonate or multielement buffer via diet or rumen: effects on performance and acid-base status of lactating cows. Our objective was to compare the influence of dietary NaHCO3 and a multielement buffer on ruminal acid-base status and lactation performance of dairy cows. Five ruminally fistulated, primiparous and multiparous lactating Holstein cows averaging 123 +/- 21 d postpartum were assigned randomly to treatments in a 5 x 5 Latin square with 3-wk experimental periods. Treatments were a basal diet without supplemental buffers, with 1.5% NaHCO3 or 1.5% multielement buffer, or with NaHCO3 or multielement buffer solutions poured into the rumen via cannula at 2 h postfeeding. Addition of either buffer to the diet reduced ruminal fluid hydrogen ion concentration from 0 to 6 h postfeeding; only NaHCO3 reduced ruminal fluid acidity when dosed via the cannula. Addition of buffers via ruminal cannula appeared to retard the reduction in ruminal fluid acidity that normally occurs from 6 to 12 h postfeeding; this may have been related to a feedback mechanism inhibiting salivary buffer secretion. Buffering capacity of ruminal fluid tended to increase with buffer addition; the increase was greatest during infusion of NaHCO3. The ruminal fluid buffer value index increased by 4 units for control cows from early (0 to 6 h) to late (6 to 12) postfeeding; smaller increases were noted for addition of multielement buffer. This index was not different for NaHCO3 during these two intervals. Milk yield and DMI were not affected by buffer addition. Although milk fat content tended to be higher with the multielement buffer than with NaHCO3, it was not accompanied by the expected alterations in ruminal acid-base status. Therefore, this increase may be related to systemic effects of specific minerals in the multielement buffer rather than to a more stable ruminal environment. Based on the ruminal fluid buffer value index, NaHCO3 tended to maintain the most stable ruminal acid-base status.


Direct measurement of myocardial free radical generation in an in vivo model: effects of postischemic reperfusion and treatment with human recombinant superoxide dismutase. The purpose of this study was to determine whether postischemic reperfusion of the heart in living rabbits induces a burst of oxygen free radical generation that can be attenuated by recombinant human superoxide dismutase administered at the moment of reflow. This phenomenon was previously demonstrated in crystalloid perfused, globally ischemic rabbit hearts. Thirty-two open chest rabbits were assigned to one of four groups of eight animals each: Group I (control animals), no coronary artery occlusion; Group II, 30 min of circumflex marginal coronary artery occlusion without reperfusion; Group III, 30 min of coronary occlusion followed by 60 s of reperfusion, and Group IV, 30 min of coronary occlusion followed by treatment with recombinant human superoxide dismutase (a 20-mg/kg body weight bolus 90 s before reperfusion and a 0.17-mg/kg infusion during 60 s of reperfusion). Full thickness biopsy specimens taken from the ischemic region were then rapidly freeze clamped and electron paramagnetic resonance spectroscopy was performed at 77 degrees K. Three radical signals similar to those previously identified in the isolated, crystalloid perfused rabbit heart were observed: an isotropic signal with g = 2.004 suggestive of a semiquinone, an anisotropic signal with g parallel = 2.033 and g perpendicular = 2.005 suggestive of an oxygen-centered alkyl peroxy radical, and a triplet with g = 2.000 and aN = 24 G suggestive of a nitrogen-centered radical. In addition, a fourth signal consistent with an iron-sulfur center was seen. The oxygen-centered free radical concentration during normal perfusion (Group I) was 1.8 +/- 0.8 mumol compared with 4.4 +/- 0.9 mumol after 30 min of regional ischemia without reperfusion (Group II) and 13.0 +/- 2.5 mumol after 60 s of reperfusion (Group III) (p < 0.05 among all three groups). In contrast, superoxide dismutase treated-rabbits (Group IV) demonstrated a peak oxygen radical concentration of only 5.9 +/- 1.2 mumol (p < 0.05 vs. Group III). This study demonstrates that reperfusion after regional myocardial ischemia in the intact rabbit is associated with a burst of oxygen-centered free radicals. The magnitude of this burst is greater than that seen after a comparable duration of global ischemia in the isolated, buffer-perfused rabbit heart preparation and is significantly reduced by superoxide dismutase administration begun just before reflow.


Oxysterol-induced endothelial cell dysfunction in culture. Cholesterol oxidation products (oxysterols), such as cholestan-3 beta,5 alpha,6 beta-triol (Triol), may be atherogenic by altering the barrier function of the vascular endothelium. We have shown that incubation of endothelial cell monolayers with Triol increased transendothelial albumin transfer (i.e., decreased barrier function) in a concentration- and time-dependent manner. Such dysfunction of endothelium could result from alterations in membrane characteristics, including changes in membrane-associated enzyme activities. To test this hypothesis, endothelial monolayers were treated with 20 microM Triol and the activities of selected membrane enzymes were measured at 0, 2, 4, 6, 12 and 24 hours. Calcium-adenosine triphosphatase (Ca(++)-ATPase) and sodium, potassium, magnesium-adenosine triphosphatase (Na+, K+, Mg(++)-ATPase) activities were significantly increased after 4 or 2 hours incubation with 20 microM Triol, respectively. 5'-nucleotidase activity was significantly elevated only after a 24-hour exposure to Triol, whereas there was no change in angiotensin-converting enzyme (ACE) activity in response to 20 microM Triol treatment at any time studied. Compared with all concentrations tested 40 microM Triol increased Ca(++)-ATPase activity most markedly, with a significant increase already after a 2-hour exposure. No major morphological changes were noted until 12 hours of exposure to 20 microM Triol; obvious cellular damage was observed by 24 hours. Cultures treated with Triol for 24 hours showed significant signs of toxicity, measured by an elevated [3H]adenine release, compared with control cultures. These data demonstrate that Triol alters the activity of certain membrane-bound enzymes, particularly Na+, K+, Mg(++)-ATPase and Ca(++)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)


Zinc and iron utilization in young women consuming a beef-based diet. The purpose of this study was to determine the utilization of zinc in young adult women fed diets containing varying amounts of lean ground beef. A secondary objective was to determine the utilization of iron. The 28-day study was divided into one 7-day preexperimental period, during which a vegetarian diet was fed, and three randomly arranged 7-day experimental periods. During the experimental periods, the diets contained 3, 6, or 9 oz of beef, supplying varying levels of zinc and iron. Complete collections of urine and fecal samples were obtained from each subject. All the diets resulted in positive zinc balance; however, mean zinc balances were significantly higher when subjects consumed the experimental diets containing beef. When data for all diets containing beef were combined, mean values for apparent zinc absorption and zinc retention were not significantly higher than values that resulted from the vegetarian diet. Consumption of all diets resulted in a positive iron balance; however, mean iron balances were significantly higher for diets containing beef. As the amount of beef in the diet increased, the bioavailability of iron increased. We found that zinc and iron utilization was enhanced after consumption of a diet including lean ground beef. Results of this study also suggest that little increase in zinc and iron utilization is realized by including more than 3 oz beef per day in the diet.


The pathological effects of glove and condom dusting powders. Dusting powders are used on latex gloves and condoms. They help to release these products from their moulds during manufacture and facilitate processing and donning. Dusting powders are also used on contraceptive diaphragms, sanitary napkins and in toiletries. There are numerous dusting powders in current use around the world. At present a consensus exists about the best dusting powder to use on surgical gloves, based on data about the safety of these agents in humans, as well as their manufacturing qualities. No such consensus exists for the use of dusting powders in other situations. However, there is uncertainty about whether dusting powders used in situations other than on surgical gloves are responsible for harmful effects. This study uses intraperitoneal injections in rats to examine the pathological effects of dusting powders that are in current use on surgical gloves and condoms. We suggest that dusting powders may be a source of morbidity when used in situations other than on surgical gloves and confirm the findings of previous investigators that the dusting powders we tested differ in their ability to produce pathological effects.


Cytoskeletal features of alveolar myofibroblasts and pericytes in normal human and rat lung. Frozen or paraffin-embedded human and rat lung specimens were stained with antibodies against total actin, alpha-smooth muscle (SM) actin, vimentin, desmin, or gelsolin. Alveolar interstitial myofibroblasts [i.e., contractile interstitial cells (CIC)] were labeled by total actin antibody but not by alpha-SM actin antibody. They stained for vimentin and gelsolin and, in rat lungs, most of them for desmin. Pericytes located around venules at the junction of three alveolar septa were always positive for alpha-SM actin and never for desmin. Tissue samples were also immunostained by an alpha-SM actin antibody and studied by electron microscopy. With this technique we confirmed that cells, identified as pericytes on the basis of their location, were intensely labeled by alpha-SM actin antibodies, whereas alveolar myofibroblasts were not. We conclude that in the lung interstitium pericytes and alveolar myofibroblasts have distinct cytoskeletal features, alpha-SM actin antibody staining being a simple method to distinguish between them. Furthermore, it appears that alveolar myofibroblasts have a peculiar pattern of cytoskeletal protein composition which, in the rat, is similar to that previously described for stromal cells in uterine submucosa, liver sinusoids (Ito cells), or the core of intestinal villi.


Molecular cloning of the cDNA encoding a receptor tyrosine kinase-related molecule with a catalytic region homologous to c-met. Receptor tyrosine kinases mediate a range of growth and differentiation processes in multiple biological systems. In this work, we report the identification of a novel tyrosine kinase-related molecule, nyk-r, and the molecular cloning of its complete cDNA. Its extra-cellular domain bears no apparent homology with other receptor families, but its intracellular kinase-related region has considerable similarity with members of the insulin-receptor family such as c-met and trk B. Also, the nyk-r gene is expressed in a wide range of tissues and cell lines.


[Effect of chemotherapy on the prognosis of ovarian cancer]. Through the collaboration of 22 institutions nationwide, a total of 1,185 cases of ovarian cancer treated between January, 1980 and December, 1987, were investigated as to their prognosis from the aspect of the chemotherapeutic effect. (1) An excellent effect of the remission-induction chemotherapy was observed in a group receiving combination therapy with CDDP as the main ingredient. In particular a significant effect was seen in stages III and IV. In addition, the effect on the remaining tumors by diameter also showed a significant difference in cases of tumors of not less than 2 cm in diameter. (2) As to the effect on histological types, a comparison in stage III showed a favourable effect on endometroid, serous and mucinous adenocarcinomas, while no effect was observed in clear cell adenocarcinoma. (3) The effect of the remission induction chemotherapy did not always give rise to an improvement in the long-term prognosis of ovarian cancer, and the establishment of a therapeutic method aimed at the prevention of recurrence was desired. (4) To improve the long-term prognosis, intermittent (or cyclic) chemotherapy with CDDP as the main ingredient was found to be very effective, but maintenance chemotherapy with orally administered of 5-Fluorouracil or Tegaful was not effective. (5) The effect of the conventional immunotherapy was not observed at all.


[Study on the management of dysplasia of the uterine cervix]. In this study, we investigated problems on the management of dysplasia on the basis of our clinical data obtained in the past ten years. In addition, we also examined the biological significance of protooncogene expression and human papilloma virus (HPV) infection in the initiation and promotion of dysplasia. Among 540 cases of dysplasia we have managed, 48.8% of the cases of mild, moderate and severe dysplasia which we followed up for more than 6 months regressed, and 24.1% of the cases progressed. The cure rate for laser therapy based on the follow up for 6 to 96 months was significantly high (97.9%) in 342 cases treated by the cone method, and low (89.5%) in 38 cases treated by the vaporization method. Preoperative histological findings confirmed 60.5% of postoperative findings, and several early cervical carcinomas were found in preoperative cases of dysplasia by laser conization. Among 28 cases (8.2%) of incomplete excision, 24 cases (85.7%) regressed spontaneously. On the other hand, the positive rate of immunostaining of epidermal growth factor receptor (EGF-R) and c-myc oncogene product (c-myc protein) increased from mild or moderate to severe dysplasia, and the positive rate for EGF-R was significantly high (80.0%) in the progressive group. The positive rate for HPV genome was quite low (16.0%) in dysplasia. Among them, EGF-R was most associated with the prognosis of dysplasia. These results suggest that laser conization should be performed for many cases of dysplasia because of the preoperative possibility of the existence of early cancer, and EGF-R is also useful in determining the necessity for therapy for dysplasia.


Involvement of prejunctional alpha 2-adrenoceptor in bovine ciliary muscle movement. The effects of adrenergic agents on bovine ciliary muscle were studied in vitro. Norepinephrine, phenylephrine, or isoproterenol did not affect the muscle tone. High concentrations of guanethidine (10(-5) M) partly inhibited the nerve-mediated contractions of the muscle. The nerve-mediated contractions also were inhibited in the following order: norepinephrine > epinephrine >> isoproterenol. The inhibition was blocked by yohimbine or phentolamine, but not by optimum concentrations of prazosin, moxisylyte, propranolol, and carteolol. Usual concentrations of phosphodiesterase inhibitors markedly inhibited the nerve-mediated and carbachol-induced contractions. These results suggest that an adrenergic alpha 2 receptor located at the nerve terminals inhibits the contraction of cholinergically innervated ciliary muscle. Adrenergic innervation seems to play an inhibitory role in the muscle tissue when the muscle tone is elevated.


Effect of low neutral endopeptidase expression on response to fMLP. The effects of the low neutral endopeptidase (24.11/CD10) exhibited by cord blood neutrophils on response to the peptide mediator of cell function f-met-leu-phe (fMLP) were investigated. Oxidative radical release (superoxide and hydrogen peroxide) and chemotactic responses to fMLP were determined and compared to the responses of normal adult neutrophils. The effect of fMLP on CD10 expression as measured by flow cytometry also was evaluated. The data show that cord blood neutrophils produce increased amounts of O2- and H2O2 largely because of a prolonged reaction time to fMLP. In addition, adult polymorphonuclear neutrophil leukocytes increase the intensity of their expression of CD10 following fMLP stimulation, whereas cord blood CD10 expression does not change. Evaluation of chemotaxis demonstrated that cord blood neutrophils exhibited a shift in the fMLP dose-response relationship showing relatively better chemotaxis to lower concentrations. In support of this observation, the inhibition of endopeptidase on adult polymorphonuclear neutrophils leukocytes by phosphoramidon was associated with an augmentation of chemotaxis to 10(-9) and 10(-10) mol/L fMLP. These studies demonstrate that cord blood and adult neutrophils respond differently to fMLP and suggest that membrane endopeptidase plays a role in the observed response patterns. The low level of expression of CD10 on cord blood neutrophils and the failure to increase its expression after fMLP stimulation suggests that adult neutrophils have preformed intracellular CD10 that is not present in the newborn. We propose that the lack of endopeptidase on cord blood neutrophils together with other known features of immaturity may play a role in the overall compromised host defense exhibited by the newborn.


Nitric oxide hemoglobin in patients receiving nitroglycerin as detected by electron paramagnetic resonance spectroscopy. Blood specimens were obtained from 30 adult patients admitted to a coronary care unit after the decision to use nitroglycerin had been made by their physicians. The samples were drawn before nitroglycerin administration, within 1 hour after starting nitroglycerin, after several hours of therapy, and more than 4 hours after discontinuing therapy. One patient was admitted twice, accounting for 31 sets of blood specimens. A positive identification of nitric oxide-hemoglobin (NOHb), with electron paramagnetic resonance (EPR) spectroscopy could be made in the blood of 10 of the subjects after they had been receiving nitroglycerin for several hours (third blood sample). In seven subjects this third blood sample was not drawn, and they were dropped from the study. A final positive finding of NOHb was made in 10 of 24 patients. NOHb has not been identified previously in human subjects given nitroglycerin, and a significant dose-response relationship was observed between nitroglycerin and NOHb. We ascribe our inability to detect NOHb in all subjects before nitroglycerin (basal levels) and after nitroglycerin in 14 subjects to concentrations that were below the limits of detection of the technique as used. Subtraction of the EPR signal for plasma ceruloplasmin was necessary to detect the NOHb EPR signals. Thus we have shown EPR spectroscopy to be a highly specific and sensitive method for detecting and quantifying NOHb in human subjects. Further refinements in the technique to improve sensitivity are possible.(ABSTRACT TRUNCATED AT 250 WORDS)


Interleukin 1-stimulated prostacyclin synthesis in endothelium: lack of phospholipase C, phospholipase D, or protein kinase C involvement in early signal transduction. The cascade of transmembrane signaling events that follow the occupancy of the interleukin 1 receptor remain poorly defined. We examined potential postreceptor transduction systems involved in human recombinant interleukin 1-beta-stimulated prostacyclin synthesis in human umbilical vein endothelium. Challenge of human umbilical vein endothelium monolayers with recombinant interleukin 1-beta resulted in dose- and time-dependent tritiated arachidonate release and prostacyclin synthesis consistent with phospholipase A2 activation. Prostacyclin synthesis after interleukin 1-beta (10 ng/ml) was detected 4 hours after stimulation and peaked at 16 to 24 hours. To examine whether interleukin 1-beta produced early activation of a phosphoinositide-specific phospholipase C, human umbilical vein endothelium monolayers were labeled with tritiated-2-myoinositol and inositol polyphosphates recovered after interleukin 1-beta stimulation. In contrast to the potent agonist, alpha-thrombin, interleukin 1-beta failed to significantly increase inositol phosphate production when examined for up to 4 hours. The absence of a significant increase in the Cai++ secretagogue, IP3, was confirmed in human umbilical vein endothelium monolayers loaded with the Ca++ photoprotein probe aequorin. Basal aequorin luminescence was unaltered after interleukin 1-beta (0 to 2 hours), whereas both alpha-thrombin and Ca++ ionophore A23187 produced rapid rises in Cai++. The intracellular Ca++ antagonist BAPTA and the extracellular Ca++ chelator EGTA produced significant inhibition of interleukin 1-beta-stimulated prostacyclin generation at 4 to 8 hours, suggesting either an indirect inhibitory effect of these agents on phospholipase A2 activity or that an increase in Ca++ may be a late event in the transduction scheme after interleukin 1 stimulation. Interleukin 1-beta-stimulated protein kinase C, phospholipase D, and adenylyl cyclase activities (0 to 4 hours) were unchanged from controls. Despite the absence of increased plasma membrane protein kinase C activity up to 4 hours after interleukin 1, pretreatment of human umbilical vein endothelium monolayers with staurosporine or phorbol myristate acetate (18 hours) to reduce protein kinase C activities, significantly attenuated the interleukin 1-stimulated prostanoid responses at 16 hours but not at 4 hours. Furthermore, short (5 minute) pretreatment with phorbol myristate acetate dramatically augmented interleukin 1-mediated prostacyclin responses in synergistic fashion, suggesting that protein kinase C may modulate interleukin 1 signal transducing pathways. In summary, these studies suggest that interleukin 1-beta-mediated endothelial cell phospholipase A2 activity and prostacyclin synthesis occur via a novel transducing pathway that does not involve early activation of phospholipase C, phospholipase D, or adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)


In vivo effects of the steroid analogue RU486 on some aspects of intermediary and thyroid metabolism of brook charr, Salvelinus fontinalis. The glucocorticoid analogue RU486 was administered by intraperitoneal injection to brook charr (Salvelinus fontinalis) to further explore the role of cortisol on aspects of intermediary and thyroid hormone metabolism of the species. RU486 significantly elevated the hepatosomatic index, hepatic G3PDH activity, and hepatic glycogen content, but was without effect on hepatic protein content, hepatic FPBase activity, or plasma glucose concentration. However, the stressor-related increase in plasma glucose concentration that was evident in brook charr 24 h following handling and injection was suppressed in RU486-treated groups. The distribution volume, turnover rates, and metabolic clearance rates of [3H]cortisol were similar in RU486- and vehicle-treated groups. Plasma T3 and T4 concentrations were similar in RU486- and vehicle-treated groups, but hepatic T3 production and hepatic T3 content were lower in RU486-treated fish; TSH had no effect on hepatic T3 content of vehicle-treated brook charr but significantly increased T3 content in the RU486-treated group. These observations support the concept of a role of cortisol in the control of peripheral monodeiodination of T4 in salmonid fish and suggest that RU486 may be a useful drug for evaluating the role of cortisol in fish in vivo.


Isolation and characterization of an antiviral flavonoid from Waldsteinia fragarioides. The antiviral agent in a fraction from Waldsteinia fragarioides (Rosaceae) was purified using bioassay-guided fractionation of activity against herpes simplex type 1 virus. Structural eluciation by instrumental methods identified the active component to be the known flavonoid glycoside, isoquercitrin (3,3',4',5,7-pentahydroxyflavone-3 beta-O-glucoside), which had not previously been shown to possess antiviral activity.


Phase II trial of chlorozotocin and fluorouracil in islet cell carcinoma: a Southwest Oncology Group study. A phase II trial that used fluorouracil (5-FU) and chlorozotocin (CTZ) was performed in patients with metastatic islet cell carcinoma to determine the response rate and toxicity. Patients received four cycles of induction chemotherapy. Good-risk patients received 5-FU 800 mg/m2/d days 1 to 4 as a continuous intravenous (IV) infusion (CIV) and CTZ 175 mg/m2 IV on day 1. Poor-risk patients (previous radiation to > or = 25% bone marrow-bearing areas; serum bilirubin > or = 5 mg/dL; creatinine > 1.0 mg/dL) received 5-FU 600 mg/m2/d and CTZ 75 mg/m2 in a similar manner. In responding or stable patients, reduced doses of 5-FU and CTZ were continued as maintenance therapy (maximum, 18 months). Forty-seven of 51 patients were eligible, and 44 received chemotherapy. Fourteen of 44 patients had partial responses, with 13 of 36 (36%; 95% confidence interval [CI], 21.0% to 54.0%) good-risk patients and one of eight (12%; 95% CI, 0.3 to 52.6%) poor-risk patients responding. Median survival of all patients was 25 months, and the median response duration was 11 months. Side effects were moderate to severe and included myelosuppression and gastrointestinal toxicity. Thirteen patients developed renal toxicity, which was severe or life-threatening in five. This seemed to be related to the administration of cumulative doses of CTZ > or = 1,500 mg. These results demonstrate that the combination of 5-FU and CTZ has activity in islet cell carcinoma, but the occurrence of renal toxicity secondary to CTZ may limit the use of this agent.


Adjuvant chemotherapy after radical surgery for non-small-cell lung cancer: a randomized study. The aims of this study were to assess the effect of adjuvant chemotherapy on overall survival, disease-free survival, and relapse pattern, as well as its toxicity in patients who underwent radical surgery for non-small-cell lung cancer (NSCLC). One hundred ten patients with T1-3N0 (World Health Organization [WHO] 1981) NSCLC underwent radical surgery during the period of 1982 through 1987. After surgery, the patients were randomized to receive adjuvant chemotherapy (n = 54) (cyclophosphamide 400 mg/m2, doxorubicin 40 mg/m2, and cisplatin 40 mg/m2 [CAP] for six cycles) or no active treatment (n = 56). After 10 years from the start of the study, 61% of patients were alive in the chemotherapy group and 48% were alive in the control group (P = .050). Seventeen patients (31%) in the CAP group and 27 patients (48%) in the control group had a recurrence during the follow-up period (P = .01). The 5-year survival rate was 67% in the chemotherapy group and was 56% in the control group (P = .050). The patients in the chemotherapy group who completed the planned treatment had a slightly better 5-year survival than those whose chemotherapy was discontinued (72.5% v 50.3%; P = .15). Chemotherapy-related gastrointestinal toxicity grade 3 to 4 (WHO) occurred in 63% and was the main reason why patients refused further planned therapy. Our results suggest that patients with NSCLC at pathologic stage I who have undergone radical surgery benefit from adjuvant chemotherapy.


Short-term regulation of the renal vitamin D receptor in rats by 1,25-dihydroxycholecalciferol is calcium insensitive. Acute regulation of the vitamin D receptor in kidney by 1,25-dihydroxycholecalciferol and dietary calcium was investigated using vitamin D-deficient rats. 1,25-Dihydroxycholecalciferol administered to normocalcemic, vitamin D-deficient rats increased the renal receptor level, whereas serum calcium and phosphorus concentrations remained nearly constant. In hypocalcemic, vitamin D-deficient rats, 1,25-dihydroxycholecalciferol caused a sharp response at 4 h, which remained elevated for the remaining 20 h. Serum calcium rose gradually over 24 h, whereas serum phosphorus remained fairly constant. When hypocalcemic, vitamin D-deficient rats were fed a high calcium diet following 14 h without food, the serum calcium concentration increased and serum phosphorus concentration decreased, whereas renal receptor levels were unchanged. These data indicate that 1,25-dihydroxycholecalciferol rapidly regulates the renal vitamin D receptor, independent of serum calcium and phosphorus, but requires an adequate serum calcium concentration to sustain a prolonged effect.