Characteristics of natural conceptual cycles occurring in a prospective study of sex preselection: fertility awareness symptoms, hormone levels, sperm survival, and pregnancy outcome. A prospective study of sex preselection provided the opportunity to characterize the fertile menstrual cycle. We describe from 91 natural conceptual cycles, or sub-groups thereof, cervical mucus symptoms, basal body temperature (BBT) changes, hormonal characteristics, and the outcome of pregnancy. The cervical mucus symptoms defined a potential fertile period of 10 days' average length, with the "peak" mucus symptom occurring at a mean of day 15.5 of the cycle. A fertile period of 9 to 10 days was also indicated by pregnancies resulting from single acts of intercourse between -6 and +3 days from ovulation. The BBT chart was biphasic in 73 of 76 cycles. The duration of the LH surge as observed in early morning urine samples averaged five days, with the peak occurring 1.4 days after the onset. Considerable inter-subject variability was seen in the LH excretion levels. Hormone measurements of peripheral plasma during the luteal phase showed the first detectable presence of hCG between day 7 and day 13 after conception. Progesterone concentrations in the midluteal phase exceeded 20 nmol/L and tended to be higher than in a comparison group of nonfertile cycles, whereas the estradiol concentrations were similar in fertile and nonfertile cycles. The birth sex ratio favored males when intercourse preceded ovulation/fertilization by two days or longer. While this association was statistically significant, the number of pregnancies involved is too small to conclude that the relationship is real.


Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes. Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.


CA 125 levels in monitoring therapy for endometriosis and in prediction of recurrence. CA 125 levels were measured in the serum of 18 patients with laparoscopically diagnosed stage 2 to stage 4 endometriosis (American Fertility Society classification) and in eight normally cycling control women. All endometriosis patients were treated medically with either Danazol or Buserelin. CA 125 levels were measured during treatment and during the 18-month follow-up period. The mean CA 125 level in women with endometriosis was significantly higher than that observed in normal women (28.6 +/- 5.1 (+/- SE) units/mL vs. 11.1 +/- 1.1 units/mL). However, there was considerable overlap of values between controls and patients with stage 2 disease. The levels in patients with stage 3 or stage 4 disease were always greater than 20 units/mL. There was a significant positive correlation (r = .51) between the implant score and CA 125 levels, while there was no correlation between the total score (which includes adhesions and implants) and the CA 125 levels. Four of the patients who had recurrence of symptoms approximately 1 year after treatment had CA 125 levels close to pretreatment levels, and recurrence of endometriosis was confirmed by laparoscopy. The CA 125 levels in the rest of the patients remained suppressed during the follow-up periods. These results indicate that (1) CA 125 level can predict active endometriosis lesions in patients with stage 3 and stage 4 endometriosis, but is of no value for predicting adhesions; (2) CA 125 levels are useful in monitoring therapy during treatment; (3) during the follow-up period, elevations in CA 125 might predict recurrence of disease in women with stage 3 and stage 4 endometriosis.(ABSTRACT TRUNCATED AT 250 WORDS)


Excess of taurine supplementation in rat: effects on GABA-related amino acids in developing nervous tissues. The effects of taurine supplementation on GABA-related amino acid homeostasis in developing nervous tissues of suckling rats were studied. In the first two weeks of postnatal growth, cerebral cortex and cerebellum appear more accessible to taurine supplementation in comparison to retina; in addition, different changes in excitatory/inhibitory amino acids were observed. After the 5th day of life, in the retina and cerebellum of taurine-supplemented pups a decrease in GABA levels was found; in contrast, in cerebral cortex GABA content significantly increased throughout 20 days of postnatal growth. In all nervous tissues studied (except for cerebellum) glutamine concentration increased at the 5th day; then in cerebellum and in retina, but not in cerebral cortex, a significant decrease until the 20th day occurred. Furthermore, in cerebellum and retina taurine supplementation decreased glutamate levels, in comparison to controls, at the 10th and until the 20th day of postnatal life, respectively, whereas in cerebral cortex an increase in glutamate level was observed only at the 5th day. In conclusion, taurine supplementation, in excess to the usual amount from the mother's milk, affected the glutamate compartments in various cell types. The changes in GABA-related amino acid concentrations in cerebral cortex, cerebellum, and retina may depend on the different pattern of the metabolic processes at different maturative stages.


Characterization of the bacterially expressed Drosophila engrailed homeodomain. To investigate the mechanism of DNA recognition by the homeodomain, truncated proteins containing the entire homeodomain encoded by the Drosophila engrailed gene were expressed in Escherichia coli. Each protein was accumulated to an amount representing more than 40% of the total bacterial protein and recovered in the soluble fraction. Of the three truncated proteins, the shortest one (71 amino acid residues) was further purified by conventional chromatography. The purified engrailed homeodomain (En-HD) protected a DNA sequence, TTAATT, the core element of consensus sequences recognized by many other homeodomain proteins, from DNase I digestion. UV-CD spectra of the En-HD showed that it mainly consisted of alpha-helix. Based on one-dimensional 1H-NMR spectra, the tertiary structure of the En-HD was shown to be stable against temperature up to 50 degrees C and low pH. The low pH resistance of the protein was also demonstrated by UV-CD measurement. Thus, the current over-production system provides an active and stable homeodomain, which is suitable for structure-function analysis.


Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles. The immunocytological distribution of the proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication, has been examined during the early development of Xenopus laevis. The protein is uniformly detected in nuclei during early stages up to the neurula stage. PCNA is detected by its distinctive cyclical pattern during early development, remaining detectable only during the period of S phase of each cell cycle. Immunological detection of PCNA is therefore a useful and specific non-isotopic marker of S-phase cells in the embryo. PCNA associates with typical karyomeric structures, suggesting that DNA replication starts before the nuclear compartment is entirely formed. At the midblastula transition, a new pattern of PCNA staining becomes apparent. First, a new type of PCNA staining is detected at the nuclear periphery. Second, mitotic clusters with different PCNA distributions suggest that the onset of desynchronization of the cell cycle at this stage is not random.


Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome. The SPN antigen plays an essential role in mitosis, since microinjection of antibodies causes mitotic arrest. Here we show, by examination of the relative locations of SPN antigen, the centrosomal 5051 antigen and tubulin in normal mitotic, and in taxol-treated mitotic cells, that the SPN antigen is involved in organizing the microtubules of the spindle. The 210 kDa protein defined as SPN antigen relocates from the nuclear matrix to the centrosome at prophase, remains associated with the poles at metaphase and anaphase, and dissociates from the centrosomes in telophase. In taxol-treated mitotic cells, SPN staining shows a striking redistribution while 5051 antigen remains associated with centrosomes. SPN antigen is seen at the plasma membrane end of the rearranged microtubules. SPN antigen is always at the center of the multiple microtubule asters (5 to 20 per cell) induced by taxol, whereas 5051 again remains associated with the centrosomal complex (1 to 2 foci per cell). Microtubule nucleation is associated with the SPN antigen rather than with the 5051 antigen. Microinjection of SPN-3 antibody into taxol-treated mitotic PtK2 cells causes disruption of the asters as judged by tubulin staining of the same cells. Finally, SPN antigen extracted in soluble form from synchronized mitotic HeLa cells binds to, and sediments with, pig brain microtubules stabilized by taxol. This association of SPN antigen with microtubules is partially dissociated by 0.5 M NaCl but not by 5 mM ATP. Thus SPN antigen binds to microtubules in vitro and seems to act as a microtubular minus-end organizer in mitotic cells in vivo.


Comparison of serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, and genetic restriction fragment length polymorphism analysis for identification of rickettsiae: characterization of two new rickettsial strains. In 1990, 17 adult Rhipicephalus turanicus ticks were collected in the south of France. Two spotted fever group rickettsiae, Mtu1 and Mtu5, were isolated from the hemolymphs of two of these ticks by the centrifugation shell-vial technique by using HEL cells. These isolates were compared with reference spotted fever group rickettsial serotypes by using three identification methods: microimmunofluorescence serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis. The results obtained by all these techniques showed that Mtu1 and Mtu5 are each previously undescribed rickettsial serotypes. A comparison of the three methods used to identify the isolates led us to the conclusion that, in large-scale epidemiological studies, the simplest way to identify isolates in ticks is to first use the polymerase chain reaction-restriction fragment length polymorphism analysis directly on triturated ticks as a screening method to detect interesting rickettsiae, and then attempt to isolate rickettsiae from ticks for identification by microimmunofluorescence and SDS-PAGE, both of which are time-consuming and expensive to carry out.


Ribotyping provides efficient differentiation of nosocomial Serratia marcescens isolates in a pediatric hospital. Ribotyping with a nonradioactive probing system was used for the epidemiological evaluation of 15 Serratia marcescens nosocomial strains isolated from the stools of 12 children with no apparent illness in five different hospital wards over a 20-day period. Our results indicate that the occurrence of S. marcescens colonization was the result of the spread of a single epidemiological strain in the hematology ward, the oncology ward, and the gastroenterology ward and in two neonates in the neonatology ward, suggesting cross-contamination between the patients in these four wards. This isolate was genotypically unrelated to the bacterial strain found in the three other patients in the neonatology ward. Interestingly, one patient in the neonatology ward harbored these two genotypically different strains. Finally, the patient in the intensive care unit was colonized with a different strain. We find ribotyping to be a more reliable technique than biochemical typing. The results of ribotyping are more easily interpreted than are those of total DNA analysis, with an equivalent degree of discrimination.


DNA polymorphisms in methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus. Restriction fragment length polymorphisms in methicillin-susceptible and methicillin-resistant (MRSA) strains of Staphylococcus aureus isolated in the same hospital over a 4-month period were studied by using SmaI and ApaI digestion of genomic DNA and pulsed-field gel electrophoresis. Each of the 20 methicillin-susceptible strains had a unique SmaI pattern, but the 27 MRSA strains showed only seven SmaI patterns. More than half of the SmaI fragments in all of these seven patterns were identical, as were those in the patterns from two unrelated MRSA strains. Digestion with ApaI, which cuts staphylococcus DNA into at least twice as many fragments, confirmed the results obtained with SmaI. Lastly, the plasmid contents of MRSA strains showing identical SmaI and ApaI electrophoretic patterns were not identical. These results are interpreted as supporting the hypothesis that all MRSA strains arose from a single clone and emphasize the need to use several methods in epidemiological investigations of MRSA outbreaks.


Typing of Histoplasma capsulatum by restriction fragment length polymorphisms in a nuclear gene. We previously described yps-3, a Histoplasma-specific nuclear gene probe useful in the identification of Histoplasma capsulatum. By using restriction fragment length polymorphisms (RFLPs) of DNA detected by the yps-3 gene and mitochondrial DNA, 76 clinical and soil isolates of H. capsulatum were classified. The majority of North American isolates obtained from endemic regions of the midwestern United States were members of the previously characterized class 2, although four clinical isolates from different patients with AIDS from that region were grouped in class 1 with the temperature-sensitive Downs strain. A Florida soil isolate (FLS1) was placed in class 4 on the basis of RFLP with both probes. Two American Type Culture Collection strains (G184B and G186B) from Panama were grouped into class 3 by this analysis. A group of five H. capsulatum isolates obtained from patients with AIDS in New York City were typed into a new class 5 on the basis of yps-3 polymorphisms; those organisms fell into two broad mitochondrial DNA patterns, designated 5b and 5c. Two new isolates from Panama were also members of this broad yps-3 class 5 group, but they exhibited a distinct mitochondrial DNA profile (class 5a). A sixth class was detected in DNA obtained from a patient with AIDS from Panama; that DNA had unique RFLP profiles with respect to both probes. These observations suggest that the Histoplasma-specific yps-3 gene probe is a sensitive tool for typing H. capsulatum in clinical specimens. Additionally, these studies provide molecular support for the hypothesis that AIDS-associated histoplasmosis in nonendemic areas is due to reactivation of a previously acquired infection.


Inhibitory effect of cetirizine on the bronchial eosinophil recruitment induced by allergen inhalation challenge in allergic patients with asthma. In patients with asthma there is a recruitment of eosinophils in bronchoalveolar lavage fluid (BALF) after the late asthmatic reaction (LAR). Cetirizine is a selective H1 antagonist that inhibits the eosinophil recruitment induced by allergen in the skin. The aim of this study was to evaluate whether cetirizine was able to inhibit the LAR-induced inflammatory reaction. Twelve allergic asymptomatic subjects with asthma (aged 18 to 58 years) without any treatment were enrolled in the study; FEV1 was greater than 83% predicted in each case. An allergen inhalation-challenge test was performed to assess the presence of an LAR. In a double-blind, randomized, placebo-controlled study, the patients were treated for 8 days with either cetirizine, 15 mg twice a day (six patients, group 1), or placebo (six patients, group 2). On day 8, a second allergen inhalation-challenge test with the same allergen was performed, and BAL was realized 24 hours later; as usual 250 ml of saline was instilled by 50 ml aliquots, and the first recovery was analyzed separately. In each case, the LAR observed after treatment was similar to the first one. In placebo-treated patients, an increased number of cells, mainly eosinophils, was observed in the first recovery of BALF compared with the number in subsequent recoveries. These numbers were significantly higher than numbers observed in cetirizine-treated patients. Cetirizine did not modify significantly the allergen inhalation-challenge test, but it inhibited the recruitment of inflammatory cells, mainly eosinophils.


In vivo growth hormone (GH) response to human GH-releasing factor (GRF) or somatostatin (SRIF) in foetal pigs. The short-term effect of hypothalamic GRF and SRIF on the pituitary release of GH at different stages of gestation has been studied. In the present experiment eighteen gilts were used, six at each of 66, 88 and 110 days of gestation. Ventral laparotomy was performed under general anaesthesia and a section of uterus was exteriorized. Blood samples were obtained from the umbilical vein of three foetuses per gilt just prior to the injection of each foetus with either saline, 5 micrograms/kg of hGRF (1-44)NH2 or 50 micrograms/kg of SRIF into the umbilical vein. Additional blood samples were obtained 15, 30, 45 and 60 min post-injection. Serum samples were radioimmuno-assayed for GH (porcine). There was a treatment by gestational age interaction (P less than 0.01) on mean GH concentrations, area under the GH curve and GH peaks. While treatments had no effect (P greater than 0.1) on GH variables at 66 days of gestation, the area under the GH curve was slightly increased by GRF (P = 0.14) at day 88 and all GH variables were significantly increased (P less than 0.01)) by GRF at 110 days of gestation. There was a quadratic effect of time post-injection on GH concentrations at 88 (P less than 0.05) and 110 (P less than 0.001) days of gestation. There was no effect of SRIF injection (P greater than 0.1) on GH concentrations at any gestational age. In conclusion, the foetal pituitary responsiveness to GRF develops with foetal age and is maximal at the end of gestation, whereas there is no short-term response to a bolus of SRIF at any stage of gestation.


Diseases of the intestine mimicking Crohn's disease. We describe five patients who were initially thought to have Crohn's disease and were treated accordingly. The original diagnosis was based upon clinical presentation, roentgenograms, and histological examination, but subsequent follow-up showed that diagnosis to be in error. The following diagnoses were established instead: tuberculosis, Actinomyces Israeli infection, reaction to gold therapy, metastatic cancer, and linitis plastica. We stress the importance of considering conditions that can mimic Crohn's disease.


Altered IFN-gamma and IL-4 pattern lymphokine secretion in mice partially depleted of CD4 T cells by anti-CD4 monoclonal antibody. Complete elimination of CD4 cells by in vivo treatment with anti-CD4 mAb may result in B cell polyclonal activation. Additionally, mice treated with doses of anti-CD4 that eliminate half the CD4 cells produced higher anti-SRBC antibody responses than controls. This suggests that partial CD4 depletion enhances Th2-like function. To test this hypothesis we examined Th1 and Th2 lymphokine potential in mice partially depleted of CD4 cells. We measured IL-4 and IFN-gamma secretion by stimulated unfractionated spleen cells and analyzed activated, purified CD4 cells by RNA in situ hybridization to determine the percentage of IFN-gamma- or IL-4-producing cells. Unfractionated splenocytes from partially CD4-depleted mice secreted more IL-4 and less IFN-gamma than splenocytes from control mice. In situ hybridization proved that CD4 cells from partially depleted mice contained a higher percentage of IL-4 and a lower percentage of IFN-gamma-producing cells than controls. These results indicate that treatment with a dose of mAb resulting in partial CD4 depletion may permit increased Th2-like lymphokine expression. This study also provides evidence that cells committed to Th2-like function exist in vivo in mice.


In vivo induction of apoptosis in immature thymocytes by staphylococcal enterotoxin B. Staphylococcal enterotoxins are potent T cell mitogens. Recent studies have shown that the binding of these toxins to class II MHC molecules on accessory cells is essential for the stimulation of T cells which bear specific V beta segment of TCR. In the present study we show that i.v. administration of staphylococcal enterotoxin B (SEB) results in an enlargement of spleen and lymph nodes but causes thymus atrophy. Elimination of CD4+CD8+ cells predominantly accounted for the shrinkage of thymus, and the lowest level of this cell population was reached 4 days after SEB injection. Furthermore, this decrease in CD4+CD8+ cells was accompanied by a relative increase in the percentages of CD4+CD8-, CD4-CD8+ and CD4-CD8- cells, whereas their absolute numbers actually reduced on day 4. The thymus shrinkage involved apoptosis which was characterized by DNA fragmentation and morphologic changes. The depletion of Thy-1 high, TCR-alpha beta low and TCR-alpha beta intermediate cells also occurred with a kinetic correlated to the reduction of CD4+CD8+ cells. Our results further showed that the percentages of V beta 8+ cells reduced 12 h post SEB injection, increased after 2 days, and decreased again thereafter. SEB thus causes both apoptotic and stimulative effects in the thymus. Apparently, the tremendous loss of double-positive cells (greater than 90% in cell number on day 4) is not simply due to the reduction of V beta 8+ cells, the possible modulatory effect of other factors or hormones which may play a role in the cell death is discussed.


Participation of the CD27 antigen in the regulation of IL-2-activated human natural killer cells. The CD27 Ag is expressed by the majority of resting T lymphocytes and appears to play a crucial role in T cell activation. We found that some resting peripheral blood NK cells also express CD27. Furthermore, CD27 expression was up-regulated on NK cells stimulated by IL-2. The cytolytic activity of IL-2-activated, but not resting, NK cells was inhibited by an anti-CD27 mAb (anti-1A4). However, anti-1A4 did not affect conjugate formation between IL-2-activated NK cells and tumor cell targets. In contrast, anti-1A4 inhibited CD2-mediated calcium mobilization and the serine esterase activity of NK cell granules. These inhibitory effects could be mediated in part by increase in intracellular cAMP levels induced by anti-1A4. Our results suggest that the CD27 Ag plays an important role in the regulation of activated NK cells.


CD2 triggering stimulates the formation of platelet-activating factor-acether from alkyl-arachidonoyl-glycerophosphocholine in a human CD4+ T lymphocyte clone. A human CD4+ T lymphocyte clone synthesized platelet-activating factor (PAF) acether when stimulated via the CD2 pathway. PAF-acether was characterized by biochemical and biophysical properties and precursor-product relationships (alkyl-acyl-sn-glycero-3-phosphocholine (GPC)----alkyl-lyso-GPC (lyso-PAF)----PAF-acether) were demonstrated. The clone contained substantial amounts of alkyl-acyl-GPC. i) Hydrolysis of alkyl-acyl-GPC upon CD2 stimulation was evidenced: [3H]alkyl-lyso-GPC was formed from [3H]alkyl-acyl-GPC in [3H] alkyl-labeled cells; alkyl-lyso-GPC production was also bioassayed after CD2 triggering. ii) The rate of arachidonate transfer from diacyl-GPC to alkyl-acyl-GPC increased after CD2 stimulation of the [3H]arachidonate-labeled P28D T cells, demonstrating alkyl-lyso-GPC formation. iii) Comparison of the molecular species of the produced PAF-acether with those of arachidonate-containing alkyl-acyl-GPC raises the possibility that the produced PAF-acether is related to alkyl-arachidonoyl-GPC.


Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells. The role and interdependence of CD8+ and CD4+ alpha beta-T cells in the acute response after respiratory infection with the murine parainfluenza type 1 virus, Sendai virus, has been analyzed for H-2b mice. Enrichment of CD8+ virus-specific CTL effectors in the lungs of immunologically intact C57BL/6 animals coincided with the clearance of the virus from this site by day 10 after infection. Removal of the CD4+ T cells by in vivo mAb treatment did not affect appreciably either the recruitment of CD8+ T cells to the infected lung, or their development into virus-specific cytotoxic effectors. In contrast, depletion of the CD8+ subset delayed virus clearance, although most mice survived the infection. Transgenic H-2b F3 mice homozygous (-/-) for a beta 2 microglobulin (beta 2-m) gene disruption, which lack both class I MHC glycoproteins and mature CD8+ alpha beta-T cells, showed a comparable, delayed clearance of Sendai virus from the lung. Virus-specific, class II MHC-restricted CTL were demonstrated in both freshly isolated bronchoalveolar lavage populations and cultured lymph node and spleen tissue from the beta 2-m (-/-) transgenics. Treatment of the beta 2-m (-/-) mice with the mAb to CD4 led to delayed virus clearance and death, which was also the case for normal mice that were depleted simultaneously of the CD4+ and CD8+ subsets. These results indicate that, although classical class I MHC-restricted CD8+ cytotoxic T cells normally play a dominant role in the recovery of mice acutely infected with Sendai virus, alternative mechanisms involving CD4+ T cells exist and can compensate, in time, for the loss of CD8+ T cell function.


Divalent cation substitution reveals CD18- and very late antigen-dependent pathways that mediate human neutrophil adherence to fibronectin. We investigated the mechanisms by which Mn2+ alters human neutrophil (PMN) adherence to various connective tissue proteins. Substitution of Mn2+ for Ca2+ and Mg2+ significantly increased adhesion of human PMN to plastic well coated with fibronectin, fibrinogen, and laminin but not gelatin. Anti-CD18 mAb almost completely blocked adherence to laminin, partly blocked adherence to fibrinogen, but did not inhibit adhesion to fibronectin at all. In contrast, anti-very late antigen (VLA)-5 mAb antibodies significantly reduced Mn(2+)-mediated PMN adherence to fibronectin, but not to laminin or fibrinogen, demonstrating that VLA-5-mediated PMN adherence to fibronectin, but not to fibrinogen or laminin. This was supported by experiments in which synthetic GRGDSP peptide significantly inhibited Mn(2+)-mediated adherence to fibronectin, but not to laminin or fibrinogen. Activation of PMN with phorbol ester or C5a stimulated VLA-5-mediated adhesion to fibronectin, but the contribution of VLA-5 to the forces mediating adherence could only be detected when CD18 function was either blocked with mAb, or when CD18 was congenitally absent. VLA-5 mediated adhesion was also more transient than CD18-dependent adhesion. These data further confirm the presence of PMN VLA integrins and demonstrate that PMN VLA-5 contributes to stimulated PMN adherence to fibronectin.


IL-4 controls the selective endothelium-driven transmigration of eosinophils from allergic individuals. The mechanism leading to selective accumulation of eosinophils in allergic inflamed tissue is still unknown. In this article, transendothelial migration of circulating eosinophils from normal and allergic individuals is characterized by means of human umbilical vein endothelial cells cultivated on extracellular matrix from human fibroblasts. IL-4 pretreatment of these vascular constructs induced adherence and impressive layer penetration of eosinophils but not of neutrophils. For layer penetration, blood eosinophils from nonallergic donors needed in vitro priming by granulocyte/macrophage-CSF, IL-3, or IL-5. In contrast, freshly isolated blood eosinophils from a group of patients with atopic dermatitis spontaneously penetrated IL-4-activated vascular constructs. The here described selective pathway of eosinophil transmigration was 1) specifically induced by IL-4; 2) inhibited by the IL-4 specific, neutralizing mAb 8F12; and 3) dependent upon endothelial mRNA synthesis. Both eosinophil adherence and transmigration were present at an IL-4 concentration of 1 U/ml. The effect of endothelial preincubation with IL-4 culminated at 16 h and persisted up to 48 h. A linear increase of subendothelial accumulating eosinophils was observed within 2 h, reaching almost 100% after 4 h of coincubation. From inhibition experiments using different mAb, we conclude that the integrins CD11a/CD18, CD11b/CD18, and very late Ag-4 (CDw49d/CD29) are involved in this selective pathway of eosinophil transmigration. Taken together, this study demonstrates a novel mechanism which allows in vitro or in vivo primed eosinophils to leave the vascular compartment without influencing emigration of neutrophils.


Differentiation-related changes in quantitative binding of immunomagnetic beads. Experiments have been carried out to demonstrate that cell viability and phenotypic characteristics are not affected by exposure to constant magnetic fields or growth in the presence of Dynabeads. This paper also demonstrates that the changes in the surface concentration of the antigens, SSEA-1 (stage-specific embryonic antigen-1) and histocompatibility antigen (MHC H2-Db) during differentiation as determined by flow cytometric analysis, are mirrored by changes in the numbers of bound immunomagnetic particles (Dynabeads) targeted with monoclonal antibodies to these cell surface antigens. These results clearly indicate that the numbers of beads bound to cells reflect the numbers of specific surface antigens present on the cells.


Activation of human immunodeficiency virus by herpes simplex virus. Heterologous viruses have been examined for their ability to accelerate the course of infection with the human immunodeficiency virus (HIV) type 1. In this study, ACH-2 cells persistently infected with HIV-1 exhibited augmented HIV-1 replication as a result of superinfection with herpes simplex virus (HSV) type 1. Using HSV-1 mutants with deletions in the genes encoding immediate-early proteins ICP0, ICP4, and ICP27, it was found that ICP0 and ICP27, but not ICP4, were essential for up-regulation of HIV replication. Northern blot analysis showed that this activation of HIV was characterized by an initial rise in the level of the small, subgenomic (2.0 and 4.3 kb) mRNA species, followed by an increase in the level of unspliced genomic (9.2 kb) mRNA. Such a shift in transcriptional phase recapitulates the early-to-late transition seen in single-step growth curves of acute HIV-1 infection. Thus, HSV can activate HIV-1 from latency in ACH-2 cells, this activation of HIV is independent of productive HSV replication since the delta ICP4 deletion mutant is replication-incompetent, and this activation is evident as an increase in the steady-state levels of HIV transcripts.


[Role of p185c-erbB2 in endometrial cancer growth in vitro]. Human endometrial adenocarcinoma cells (Ishikawa line) constitutively express c-erbB2 coded oncoprotein p185erbB2 (p185) which is believed to be an orphan receptor for an unknown growth factor. Since we have shown that expression of p185 in primary lesions of endometrial cancer correlates well with high frequency of lymph node++ metastases and that the metastatic cells in the nodes strongly expressed p185, the role of the oncoprotein in processes of metastases was studied. Culturing the cells in the presence of 15% FCS and with monoclonal antibody to the extracellular domain of p185 (CB-1) inhibited cell growth and attenuated p185 expression on Western blotting, whereas no change occurred with the control antibody. Cells cultured without FCS achieved only approximately 1/3 growth compared to cells with FCS, and further suppression of growth was observed after adding CB-1. When cells were cultured on human term amnion, basement membrane invasion with p185 expression was observed. In nude mice, intraperitoneal seeding resulted in implant formation which was also associated with positive p185 as well as cyclin immunohistochemistry. In the two experiments, treatment of cells with CB-1 inhibited invasion or implant formation. The present study suggests that a signal through p185 receptor molecules acts as a trigger for early proliferation, and interaction with the host may enhance up-regulation of p185.


[A new method for diagnosis of amniotic fluid embolism by means of monoclonal antibody TKH-2 that recognizes mucin-type glycoprotein, a component in meconium]. Five monoclonal antibodies (moABs TKH-2, MA54, MA61, B72.3, and CC49), directed toward the O-linked mucin-type glycoprotein, showed signs of specific reactivity with human meconium. The reactivity of these moABs with meconium extract was examined by solid-phase ELISA with different native and sialidase-treated glycoproteins. All moABs react with meconium extract, whereas the reactivities of TKH-2, MA54, and MA61 are sialidase sensitive and the reactivity of TKH-2 with meconium extract was only inhibited by ovine submaxillary mucin (OSM), indicating that TKH-2 is the most sensitive and specific antibody clearly directed to the sialyl Tn antigen in meconium. The possible application of TKH-2 to diagnose amniotic fluid embolism (AFE) has been prelimiarily investigated. We demonstrated that the concentration of sialyl Tn antigen in the serum of patients with AFE was significantly increased, indicating that meconium was released into the maternal circulation. Our method for detecting sialyl Tn antigen in the serum of AFE patients is a direct way to demonstrate the release of meconium into the maternal circulation, and is a simple, rapid, non-invasive and sensitive method for the diagnosis of AFE.


[Molecular constitution of human arylsulfatase A and its alteration in cancer]. Arylsulfatase A (ASA) was purified from human placenta, ovary, and ovarian cancer tissue. The purified ASA was composed of three non-identical peptides of 58 kDa, 50 kDa, and 10 kDa on polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). The N-terminal sequence of the 58 kDa peptide was found to be identical with that of the 50 kDa on analysis with a protein sequencer. The peptide map of the 58 kDa peptide was quite similar to that of the 50 kDa except for some peaks. The 50 kDa peptide lacked the peptide from the residue Val445 to the C-terminus of the amino acid sequence deduced from the ASA cDNA. Four different peptides yielded by the 10 kDa peptide on HPLC had sequences near the C-terminal side. These results suggest that the 50 kDa peptide and the 10 kDa peptide are yielded by the same peptide as the 58 kDa peptide on proteolytic processing. Ovarian ASA showed the same pattern on SDS-PAGE as placental ASA. Ovarian cancer ASA was mainly composed of the 58 kDa band, and in addition the 50 kDa band decreased. The carbohydrate chain of ovarian cancer ASA was more resistant to endo-beta-N-acetylglucosaminidase H than that of normal ovary. These finding suggest that the processing of both the oligosaccharide chains and the protein unit is modified in ovarian cancer.


Long-term excess of endogenous calcitonin in patients with medullary thyroid carcinoma does not affect bone mineral density. In a cross-sectional study of 39 patients with medullary thyroid carcinoma (MTC), we have investigated the effects of long-term calcitonin excess on bone mineral density. Bone mineral density was measured by dual X-ray absorptiometry at the lumbar spine between the second and fourth vertebra and by single photon absorptiometry at the distal forearm. The mean observation time of each patient between diagnosis of tumour and measurement of bone mineral density was 62.4 months (range 1-158 months). The mean calcitonin serum level was 14.4 micrograms/l at the time of measurement of bone mineral density. All patients were substituted with 150-200 micrograms L-thyroxine daily. At both sites, the mean bone mineral densities of all patients with MTC were not significantly different from controls. Patients with normal calcitonin levels (below 0.2 micrograms/l) after treatment had a normal bone mineral density of the spine but significantly (P less than 0.05) reduced bone mineral density values of the forearm. This was due to the decreased body surface areas of patients in this subgroup. Patients with multiple endocrine neoplasia type IIa had significantly higher bone mineral densities. Other bone-influencing factors, such as postoperative hypoparathyroidism, calcium intake, diarrhoea, menopause, tumour stage, previous anti-tumour treatment, or thyroxine substitution dose, did not affect bone mineral density. We thus conclude that long-term excess of endogenous calcitonin in patients with MTC has no positive effect on bone mineral density.


The alpha chain gene of H-2O has an unexpected location in the major histocompatibility complex. A previously unknown major histocompatibility complex class II molecule consisting of the beta chain encoded by the H-2Ob gene and an unknown alpha chain was recently described. We now report that the alpha chain occurs in two allelic forms distinguished by charge difference. Using inbred recombinant mouse strains we were able to map the H-2Oa gene to a location between the A.TL and B10.MBR recombination points. Cosmids covering this region were used to isolate the gene. Sequence analysis revealed that the H-2Oa gene is the murine equivalent of the human HLA-DNA gene. These results indicate that the human HLA-DNA gene, the existence of which has long been known, is indeed coding for DO alpha, the alpha chain pairing with DO beta.


Evidence for an alpha/beta T cell-independent mechanism of resistance to mycobacteria. Bacillus-Calmette-Guerin causes progressive infection in severe combined immunodeficient mice, but not in nude mice or in mice depleted of CD4+ and CD8+ T cells. Depleting thymectomized mice of CD4+ T cells, or CD4+ plus CD8+ T cells, rendered them incapable of resolving Bacillus-Calmette-Guerin (BCG) infection in their lives, spleens, kidneys, and lungs. However, it did not render them incapable of stabilizing infection in the latter three organs after an initial period of BCG growth. Athymic nude mice showed a similar capacity to control BCG growth in these organs after a certain stage of infection. In contrast, congenitally severe combined immunodeficient (SCID) mice appeared to offer no resistance to BCG infection, in that the organism grew progressively in all organs of these mice and was lethal for them beginning on day 55 of infection. The results suggest that, although CD4+ T cells are important for resolving BCG infection, an alpha/beta T cell-independent mechanism of resistance can be acquired at 2-3 wk of infection that is capable of inhibiting further BCG growth in all organs except the lungs. Because this mechanism is absent from SCID mice, it is likely that it depends on the functions of gamma/delta T cells, B cells, or both types of cells. In keeping with this possibility is the additional finding that SCID mice engrafted with lymph node cells depleted of CD4+ or CD8+ T cells were capable of expressing an appreciable level of resistance against BCG infection.


Mechanism of the rejection of major histocompatibility complex class I-disparate murine skin grafts: rejection can be mediated by CD4+ cells activated by allo-class I + II antigen in CD8+ cell-depleted hosts. In the preceding article, we analyzed the immunohistochemical rejection mechanism of major histocompatibility complex (MHC) class I (H-2K)-disparate murine skin grafts, and showed that only CD8+ cells infiltrated at the site of the epithelial tissue of MHC class I-disparate graft. We also showed that perfect survival of MHC class I-disparate grafts were attained in thymectomized recipients treated with anti-Lyt-2 monoclonal antibody. In this report, we showed that these long-surviving allo-class I grafts were rejected in the absence of CD8+ cells by stimulation with allo-MHC class I + II-disparate graft as the second stimulation. Furthermore, it was immunohistochemically revealed that under that condition, a large number of CD4+ cells infiltrated into the epithelial tissue of these long-surviving class I grafts, which were going to be rejected 2-5 d after the transplantation of a second graft with MHC class I + II difference. This result directly shows that CD4+ cells are able to became effectors for the rejection of allo-MHC class I (H-2K) skin graft.


Evaluation of four deoxyribonucleic acid (DNA) extraction protocols for DNA yield and variation in restriction fragment length polymorphism (RFLP) sizes under varying gel conditions. This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.


Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis. Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.


Effect of storage conditions on restriction fragment length polymorphism (RFLP) analysis of deoxyribonucleic acid (DNA) bound to positively charged nylon membranes. The ability to generate an autoradiogram from deoxyribonucleic acid (DNA) immobilized on a positively charged nylon membrane could be compromised by the storage conditions of the membrane. HaeIII-digested human DNA was size fractionated and transferred to two types of positively charged nylon membranes. The membranes were stored at -20 degrees C, 4 degrees C, and ambient temperature and humidity for times ranging from 1 day to 13 weeks, then hybridized to variable number of tandem repeat (VNTR) probes to examine the effect of the storage conditions on the membrane-bound DNAs. It was shown that such membranes could be successfully hybridized and rehybridized if they were stored at -20 or 4 degrees C, but storage under ambient conditions reduced or eliminated the likelihood of successful hybridization.


Non-cast titanium restorations in fixed prosthodontics. The problems encountered in casting titanium in dentistry have not been completely resolved. The Procera system forms crowns by means of a combination of spark-erosion and milling. The accuracy of fit was examined before and after ceramic veneering both in vitro and in vivo. Before veneering, on conical surfaces space widths were approximately 53 microns in vitro and 69 microns in vivo. At shoulders and occlusal surfaces, spaces of about 430 microns were measured in vitro and of about 500 microns were measured in vivo. After ceramic veneering, slight increases in space widths could be observed. The metal-ceramic compound was tested by the 3-point bending test (DIN) and the bending test (ISO). The DIN test was satisfactory, but not the ISO test. It is concluded that titanium crowns processed by the Procera System are suitable for clinical usage, if the space widths at shoulders and the occlusal surface and the special requirements of tooth preparation are taken into account.


Synthesis and binding to beta-adrenergic receptors of p-aminobenzyl analogues of practolol and atenolol. The p-aminobenzyl analogues (8a and 8b, respectively) of the cardioselective beta-adrenergic receptor antagonists practolol and atenolol were prepared from the corresponding phenoxymethyloxiranes in 30 and 13% yields, respectively. The dissociation constants for the beta-adrenergic receptor were measured in membrane preparations of rat heart and lung. In membranes from the heart (which contain mostly beta 1-adrenergic receptors), the affinities of the derivatives and parent compounds were similar. By contrast, in membranes from the lung (which contain mostly beta 2-adrenergic receptors), the derivatives were more potent than the parent compounds. Thus, the cardioselectivities of the p-aminobenzyl analogues 8a and 8b were about one-sixth those of the respective parents.


Characterization of the beta adrenoceptor subtype(s) mediating the positive inotropic effects of epinine, dopamine, dobutamine, denopamine and xamoterol in isolated human right atrium. In patients with chronic heart failure cardiac beta-1 adrenoceptors are reduced, whereas beta-2 adrenoceptor changes vary depending on the etiology of the disease. Beta Adrenoceptor agonists can be used for short-term inotropic support in chronic heart failure; their clinical efficacy might depend on which beta adrenoceptor subtype(s) mediates their positive inotropic effect. Thus, the beta adrenoceptor subtype(s) involved in the positive inotropic effects of clinically used beta adrenoceptor agonists was characterized on isolated electrically driven human right atria by the use of the selective beta-1 adrenoceptor antagonist CGP 20712 A (300 nmol/l) and/or the selective beta-2 adrenoceptor antagonist ICI 118,551 (30 nmol/l). Epinine evoked positive inotropic effects through stimulation of beta-1 and beta-2 adrenoceptors to about the same degree, whereas dobutamine acted mainly at beta-1 adrenoceptors but had a significant beta-2 adrenoceptor component. Both agonists were full agonists causing the same maximum increase in contractile force (Emax) as did isoprenaline or Ca++ (Emax = 1.0). In contrast, denopamine was a partial selective beta-1 adrenoceptor agonist (Emax = 0.75-0.85). Dopamine was in the presence of uptake1-blockade (by 5 mumol/l phenoxybenzamine) a partial agonist (Emax = 0.60-0.70) acting selectively at beta-1 adrenoceptors; in the absence of uptake1-blockade, however, dopamine was a full agonist, indicating that part of its positive inotropic effect is indirect via the release of endogenous noradrenaline. Xamoterol did not exert positive inotropic effects, but concentration-dependently slightly decreased basal force of contraction.


An antagonist/partial agonist at the polyamine recognition site of the N-methyl-D-aspartate receptor that alters the properties of the glutamate recognition site. The effects of N-(3-aminopropyl)-1,10-diaminodecane (APDA10) on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex were investigated. In the presence of 100 microM glutamate and 100 microM glycine, APDA10 had biphasic effects on the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imin e (MK-801) to NMDA receptors on well washed synaptic plasma membranes. The maximal stimulation of binding by APDA10 was less than that seen with spermine. In the presence of glutamate and glycine, APDA10 attenuated the stimulatory effect of spermine and the inhibitory effect of 1,10-diaminodecane. In the nominal absence of glutamate and glycine, APDA10 had no effect on the binding of [3H]MK-801, but antagonized the stimulatory effect of spermine on the binding of [3H] MK-801. These data suggest that APDA10 acts as a mixed antagonist/partial agonist at the polyamine recognition site, and that the partial agonist properties of APDA10 are dependent on the activation state of the receptor complex. An increase in the potency of the glutamate site antagonists D-2-amino-5-phosphonovaleric acid and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid for inhibiting the binding of [3H]MK-801 was seen in the presence of APDA10. APDA10 also increased the affinity of binding of [3H]3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid to the NMDA receptor complex but had no effect on the binding of [3H]glycine. These data suggest that the polyamine APDA10 may alter the properties of the glutamate recognition site on the NMDA receptor complex.


The behavioral pharmacology of olanzapine, a novel "atypical" antipsychotic agent. Olanzapine (LY170053, 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5] benzodiazepine) is a novel "atypical" antipsychotic agent with 5-hydroxytryptamine2.dopamine D1/D2 antagonist activity and anticholinergic properties. In behavioral studies, olanzapine (1.25-10 mg/kg, p.o.) antagonizes apomorphine-induced climbing behavior in mice, demonstrating that the compound possesses D1/D2 antagonist activity in vivo. Olanzapine (0.3-20 mg/kg, p.o.) antagonizes 5-hydroxytryptophan-induced head twitches in mice at doses much lower than those required to block the climbing response, confirming that in vivo, the compound is a more potent 5-hydroxytryptamine2 antagonist than dopamine antagonist. Olanzapine (2.5-10 mg/kg, p.o.) also antagonized oxotremorine-induced tremor in mice. In a conditioned avoidance paradigm in rats, olanzapine inhibits the avoidance response with an ED50 of 4.7 mg/kg p.o; however, unlike other antipsychotic agents, catalepsy is only observed at much higher doses (ED50 39.4 mg/kg, p.o.). These data would suggest that the compound will be less likely to produce undesirable extrapyramidal symptoms. Unlike "typical" antipsychotics, olanzapine (1.25-5 mg/kg p.o.) increases responding during the conflict component of a modified Geller Seifter test, demonstrating that the compound may also possess anxiolytic activity. In another series of experiments, olanzapine (1.25 mg/kg, i.p.) produced clozapine-appropriate responding in a drug discrimination model in which animals had been trained to discriminate clozapine (5 mg/kg, i.p.) from vehicle. On the basis of these results, it would therefore be predicted that olanzapine will have an atypical profile and will be less likely to induce undesirable extrapyramidal symptoms than currently available drugs.


Parasympatholytic effects of vecuronium are mediated by nicotinic and muscarinic receptors in hearts of anesthetized dogs. We investigated the blocking effects of vecuronium and pancuronium on the negative chronotropic and dromotropic responses to stimulation of the parasympathetic nerves in the anesthetized, open-chest dog. We stimulated the intracardiac parasympathetic nerves to the SA nodal region (SAP stimulation) or to the atrioventricular nodal region (AVP stimulation). SAP stimulation or AVP stimulation selectively decreased heart rate or increased atrioventricular conduction time, respectively. Vecuronium and pancuronium inhibited the chronotropic response to SAP stimulation and the dromotropic response to AVP stimulation in a dose-dependent manner. The ID50 of each drug for the dromotropic response was less than that for the chronotropic response. The blocking effect of vecuronium on the negative cardiac responses to parasympathetic stimulation was about 10-fold less potent than that of pancuronium. These results suggest that the blocking effects of vecuronium and pancuronium on the negative chronotropic and dromotropic responses to parasympathetic stimulation differ from those of atropine in the heart. In the isolated right atrium perfused with blood from the support dog, vecuronium, injected into the external jugular vein of the support dog, dose-dependently inhibited the negative chronotropic and inotropic responses to carbachol or SAP stimulation and the negative followed by positive chronotropic and inotropic responses to nicotine. The ID50 values for carbachol, nicotine and SAP stimulation were not significantly different. These results suggest that parasympatholytic effects of vecuronium are mediated by not only muscarinic receptors but also neuronal nicotinic receptors in hearts of anesthetized dogs.


gamma-Glutamyltransferase-dependent biliary-hepatic recycling of methyl mercury in the guinea pig. After secretion into bile, glutathione (GSH) and GSH-conjugates are catabolized by gamma-glutamyltransferase (gamma-GT) and dipeptidases yielding glutamate, cysteine, glycine and cysteine S-conjugates, and these products are then partially reabsorbed from the biliary tree. Because methyl mercury is thought to be secreted into bile as a GSH- complex, it may be subject to a similar intrahepatic cycle, thus delaying its elimination. To examine this possibility guinea pigs were dosed with 203Hg-methyl mercury (10 mumol/kg i.v.), followed by a retrograde intrabiliary infusion of Krebs-Henseleit buffer (control) or acivicin (an inhibitor of gamma-GT). Acivicin increased biliary excretion of 203Hg by 41%, and GSH from 0.14 +/- 0.10 to 2.02 +/- 0.26 nmol/min.g of liver. Bile analyzed by gel filtration chromatography revealed that CH(3)203Hg-GSH accounted for most of this increased 203Hg excretion. When CH(3)203Hg-complexes of GSH, cysteine and albumin were introduced directly into the biliary tree by retrograde infusion, 203Hg recovery in bile was significantly lower than recovery of the nonabsorbable marker [14C]sucrose, ranging from 26.0 +/- 2.9% for CH(3)203Hg-cysteine to 48.7 +/- 5.1% for CH(3)203Hg-albumin and approximately 60% for [14C]sucrose. Acivicin pretreatment significantly increased 203Hg excretion into bile after retrograde infusion of CH(3)203Hg-GSH, whereas 203Hg recovery after retrograde infusion of CH(3)203Hg-cysteine remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)


Poly-L-aspartic acid does but triiodothyronine does not protect against gentamicin-induced cytotoxicity in the porcine kidney cell line LLC-PK1. This study investigated the protective effect of thyroid hormone and poly-L-aspartic acid (PAA) in an in vitro model of gentamicin nephrotoxicity. LLC-PK1 porcine renal cells were grown in Medium 199 supplemented with either fetal bovine serum or thyroid hormone-depleted fetal bovine serum. After a preincubation with or without 30 nM L-triiodothyronine for 3 days, or 0.1 mM PAA for 1 hr, cells were coincubated with 1 mM gentamicin for an additional 3 days. Determinations were made of the following indicators of cell damage and/or viability: the numbers of detached dead cells, the total lactate dehydrogenase activity and its percentage release and gamma-glutamyl transpeptidase activity. Preincubation with L-triiodothyronine did not protect from gentamicin-induced cell death but did reduce cellular accumulation of gentamicin (3.2 +/- 0.8 micrograms/mg of protein vs. 5.2 +/- 1.8 micrograms/mg of protein in controls; P less than .05). In contrast, preincubation with 0.1 mM PAA decreased gentamicin-induced cell death (gentamicin: 685 +/- 416% of control dead cells and 487 +/- 48% of control lactate dehydrogenase release; PAA + gentamicin: 164 +/- 63% of control dead cells and 257 +/- 85% of control lactate dehydrogenase release; P less than .05) but failed to attenuate inhibition by gentamicin of gamma-glutamyl transpeptidase activity (gentamicin: 69 +/- 7% of control; PAA+gentamicin: 76 +/- 3% of control) and failed to alter cellular gentamicin levels. Protection against gentamicin nephrotoxicity by L-triiodothyronine was not demonstrated in LLC-PK1 cells, indicating that its protective effect in vivo is likely due to a systemic effect of the hormone.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of the D1-dopamine agonists SKF-38393 and A-68930 in neonatal 6-hydroxydopamine-lesioned rats: behavioral effects and induction of c-fos-like immunoreactivity. Administration of the selective D1-dopamine receptor agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF-38393) to neonatal 6-hydroxydopamine-lesioned rats results in profound behavioral manifestations and induction of striatal c-fos-like immunoreactivity. The full D1-dopamine agonist I,[R,S]1-aminomethyl-3,4-dihydro-5,6-dihydroxy-3-phenyl-1H-2-benzopyran hydrochloride (A-68930), like SKF-38393, produced a dose-dependent, D1-selective increase in locomotor activity and striatal c-fos-like immunoreactivity. These responses were antagonized by a D1-dopamine antagonist, but not by a D2-dopamine antagonist. A-68930 produced locomotor activation at a lower dose than SKF-38393, but no dose of A-68930 was able to produce the magnitude of locomotor activation seen with SKF-38393. Both A-68930 and SKF-38393 induced similar stereotyped behaviors and possessed similar propensities to induce self-injurious behavior in neonatally lesioned rats; however, A-68930 was significantly more potent than SKF-38393 in inducing these behaviors. When either SKF-38393 or A-68930 were administered repeatedly at 2-week intervals, behavioral sensitization (priming) occurred. However, unlike SKF-38393, a high dose of A-68930 produced seizure activity and markedly desensitized D1-dopamine receptor activation for up to 3 days after administration. These results with A-68930 provide additional evidence that the specific behavioral and biochemical responses observed in neonatally lesioned rats after SKF-38393 administration are due to actions on D1-dopamine receptors, and indicate that A-68930 provides a new tool for investigating D1-dopamine receptor function.


Excitatory and inhibitory synaptic currents and receptors in rat medial septal neurones. 1. A thin-slice preparation was used to study the postsynaptic potentials and the underlying currents of visually identified rat medial septal (MS) neurones under tight-seal voltage- and current-clamp conditions. 2. Upon stimulation of the afferent fibres, all MS neurones exhibited a sequence of excitatory-inhibitory postsynaptic potentials (EPSP-IPSP). Under voltage clamp, with potassium glutamate as internal solution and at negative holding potentials (Vh), this synaptic pattern appeared as an initial inward current followed by a longer lasting outward current. 3. The inward postsynaptic current was completely abolished by 5 microM-6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) whereas the outward current disappeared in the presence of 10 microM-bicuculline. Thus the major excitatory and inhibitory synaptic inputs were identified as being due to activation of quisqualate/kainate glutamatergic and gamma-aminobutyric acid (GABAA) receptors, respectively. 4. At positive Vh a CNQX-resistant component of the excitatory postsynaptic current (EPSC) was revealed. This component was slower than the one mediated by the quisqualate receptor and was abolished by 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP), indicating that N-methyl-D-aspartate (NMDA) receptors are involved in excitatory synaptic transmission in MS cells. The existence of the two main subtypes (NMDA and non-NMDA) of glutamatergic receptors in MS neurones was also confirmed by the responses of the neurones to bath application of the different agonists (glutamate, quisqualate, kainate and NMDA). 5. The CNQX-sensitive EPSC had a reversal potential near 0 mV. The fast rise time (approximately 0.7 ms) indicates a somatic location of the excitatory synapses. The relaxation kinetics of the fast EPSC were fitted by a single exponential function with a time constant of 1.13 +/- 0.1 ms. This parameter was independent of Vh. Fast EPSCs were blocked by CNQX in a dose-dependent manner (dissociation constant, KD = 0.2 microM). 6. Inhibitory postsynaptic currents (IPSCs) were studied in symmetrical chloride solutions after blockade of the excitatory receptors. The current-voltage relation was linear and reversed at 0 mV. The IPSCs had a fast rise time and their decay was best fitted by the sum of two exponentials with time constant of approximately 20 and 50 ms (Vh = -60 mV). The IPSCs were abolished by bicuculline (KD = 1 microM), a selective antagonist of GABAA receptors. As expected, bath application of GABA produced large whole-cell currents. 7. In many cells, in addition to the usual EPSP-IPSP sequence, failures of either the EPSP or the IPSP were frequently observed during the experimental protocol.(ABSTRACT TRUNCATED AT 400 WORDS)


Potassium channels opened by noradrenaline and other transmitters in excised membrane patches of guinea-pig submucosal neurones. 1. Unitary potassium currents were recorded in outside-out patches of membrane from guinea-pig submucosal neurones. The actions of alpha 2-adrenoceptor agonists, somatostatin and [Met5]enkephalin were studied. 2. Three main groups of background potassium channels were active. At -70 mV with 160 mM-potassium on both sides of the membrane, they had conductances of 30-65 (small), 120-160 (intermediate) and 220-260 pS (large). 3. The open channel current-voltage relation showed only constant-field rectification. Extracellular barium (2 mM) and caesium (2 mM) decreased inward but not outward currents. Tetraethylammonium (10 mM) had no effect. 4. Noradrenaline, somatostatin and [Met5]enkephalin each increased the open probability of all three classes of channel when two or more unitary amplitude channels were active in the membrane patch. Agonists were ineffective when no channel, or a single channel, was discernible in the patch. Agonists did not cause the appearance of unitary currents distinct from those seen prior to their application. 5. The effect of the agonists required intracellular guanosine 5'-triphosphate. 6. The results show that the hyperpolarization of submucosal plexus neurones by noradrenaline, somatostatin and [Met5]enkephalin results from the increased opening of at least three types of background potassium channel, and that the coupling from the receptors to the channels is maintained in excised membrane patches.


An improved technique for processing an overdenture on implants. This article shows an alternate procedure for making an overdenture on implants. This method helps to eliminate a chairside reline for the dentist by directly incorporating the clips into the heat-cured acrylic resin. The procedure is divided into three main sections. The first section deals with the impressions, casts, and records. The second section relates to the making of the bar, and the third section shows the assembling of the clips.


Effect of benzoyl peroxide on two-stage oral carcinogenesis and gamma-glutamyl transpeptidase in hamsters. Hamster buccal pouches were treated with 7,12-dimethylbenz(a)anthracene (DMBA) triweekly for 3 wk and subsequently with 40% benzoyl peroxide (BP) in acetone for up to 27 wk. BP treatment resulted in a marked hyperplasiogenic effect and a weak tumor promoting effect. Whereas most carcinogens and tumor promoters induce gamma-glutamyl transpeptidase (GGT) activity, BP diminished its activity as compared to controls. Comparable results have also been noted in the liver, where a group of newly isolated hepatocarcinogens, peroxisome proliferators (PP), also characteristically deplete the GGT activity and placental glutathione S-transferase (GST-P), another tumor marker.


Bar-retained overdentures for implants--technical aspects. The principles of the laboratory procedures for making overdentures retained by bar abutments on ITI implant fixtures are described, and details of producing the master casts, making a soldered bar, and processing a prosthesis incorporating a cobalt-chromium alloy strengthener are given.


Oxygen isosteric derivatives of 3-(3-hydroxyphenyl)-N-n-propylpiperidine. Some substituted 3-phenylmorpholines (10a-e,j,k) and 3-thienylmorpholines (10f,g), isosteres of 3-(3-hydroxy-phenyl)-N-n-propylpiperidine (3-PPP), were prepared and submitted to binding assays on D-2 dopaminergic and 5-HT1 and 5-HT2 serotonergic receptors, in comparison with 3-PPP and its analogue 3a,b. The results show the loss of D-2 affinity for all morpholines, while a certain activity was still observable for piperidine derivatives. Regarding the serotonergic affinity, only chloro and methoxy derivatives (10a-d) were moderately active on the 5-HT1A receptor, either when the substituent was in the C-2 or C-3 position, whereas no tested compounds showed affinity toward the 5-HT2 receptor.


Phylogenetic analysis of the thiolase family. Implications for the evolutionary origin of peroxisomes. The thiolase family is a widespread group of proteins present in prokaryotes and three cellular compartments of eukaryotes. This fact makes this family interesting in order to study the evolutionary process of eukaryotes. Using the sequence of peroxisomal thiolase from Saccharomyces cerevisiae recently obtained by us and the other known thiolase sequences, a phylogenetic analysis has been carried out. It shows that all these proteins derived from a primitive enzyme, present in the common ancestor of eubacteria and eukaryotes, which evolved into different specialized thiolases confined to various cell compartments. The evolutionary tree obtained is compatible with the endosymbiotic theory for the origin of peroxisomes.


Regulated adenovirus mRNA 3'-end formation in a coupled in vitro transcription-processing system. The adenovirus major late transcription unit encodes five poly(A) sites whose use during infection is regulated. Early in the infection, the 5'-most site, L1, is used preferentially, whereas late in infection, all sites are used equivalently. Previous in vivo experiments indicated that regulatory sequences flank the AAUAAA and GU-rich elements of the L1 poly(A) site. We have developed an in vitro coupled transcription-processing system for studying the function of these regulatory sequences in HeLa cell nuclear extracts. The in vitro analysis using this system shows that predominant use of the L1 poly(A) site, as mediated by the upstream regulatory sequence, is independent of transcription. Furthermore, the reaction conditions are favorable to both 3'-end processing and splicing, making this system generally useful for the study of posttranscriptional processes.


[Study on adrenergic acid secretion using isolated parietal cells of guinea-pigs]. In order to investigate the effect of adrenergic nerve system on acid secretion at the cell-levels, acid secretion of isolated parietal cells stimulated by various adrenergic agonists was observed by measuring the accumulation of 14C-aminopyrine (A.P.). Both isoproterenol, beta receptor agonist, and salbutamol, beta-2 receptor selective agonist, increased A.P. accumulation, whereas dobutamine, beta-1 receptor selective agonist, showed no effect. And beta-2 receptor on parietal cells was detected by binding assay. These data may suggest that parietal cells have beta-2 receptors through which acid secretion will occur.


Cell surface aminopeptidase A and N activities in human glomerular epithelial cells. Cell surface aminopeptidases N (APN) and A (APA) have been characterized on cultured human glomerular epithelial cells and a SV40-transformed cell line derived from them. APN had a wide substrate specificity whereas APA only attacked peptides with an acidic N terminal amino acid. Both enzymes also differed by their sensitivity to divalent cations and to aminopeptidase inhibitors. Phorbolmyristate acetate (PMA) stimulated APN but not APA expression after a lag time of 12 hours. An increase of twice the basal value was observed with 10 ng.ml-1 PMA. This effect was confirmed by immunofluorescence staining using a specific anti-APN monoclonal antibody. Both ecto- and total enzyme activities were stimulated by PMA. The effect of PMA was suppressed by H7, a PKC inhibitor, and cycloheximide, an inhibitor of protein synthesis. Thrombin (1 to 2.5 U.ml-1) and interferon (IFN)-gamma (100 U.ml-1) also stimulated APN activity, the latter after longer exposure of the cells. APA activity was increased by 8-bromo-cAMP and two cAMP-stimulating agents, forskolin and isobutylmethylxanthine (IBMX). A twofold increase above basal value was obtained with 100 microM forskolin after 72 hours of treatment. cAMP-stimulated APA activity was suppressed by cycloheximide. Dexamethasone also stimulated APA activity. The effects of forskolin and dexamethasone were additive. These results demonstrate that APN and APA in glomerular epithelial cells are under different regulations: mitogens and IFN-gamma for APN, cAMP and glucocorticoids for APA. This selective expression may imply possible functional consequences in glomerular diseases.


Tetranectin, a plasminogen kringle 4-binding protein. Cloning and gene expression pattern in human colon cancer. Tetranectin is a recently discovered protein that binds to kringle 4 region of plasminogen (Clemmensen I, Petersen LC, Kluft C. Eur J Biochem 1986; 156:327. The mRNA encoding human tetranectin was cloned by using degenerate primers in a reverse transcriptase reaction followed by polymerase chain reaction amplification. The resulting polymerase chain reaction product was examined by DNA sequencing and subsequently used as probe for screening a human placental cDNA library. A full length cDNA clone (TET-1) was isolated, characterized, and used for Northern blot and in situ hybridization. DNA sequencing analysis revealed a 874-base pair cDNA containing an open reading frame of 606 base pairs encoding 202 amino acids. A classical signal peptide was present starting with the initiation methionine. The mature tetranectin chain consisted of 181 amino acids (M(r) = 20,169). The 3' noncoding region contained a single polyadenylation signal and a 26-residue poly A tail. The predicted amino acid sequence of the mature tetranectin chain showed, except for one amino acid, complete identity to that obtained by sequencing of the native protein (Fuhlendorff J, Clemmensen I, Magnusson S, Biochemistry 1987;26:6757). Northern blot of poly A+ revealed a single band of approximately 1 kb. Northern blot analysis of poly A+ isolated from a series of normal human tissues (lung, liver, spleen, kidney, and pancreas) revealed a distinct hybridization band that was especially prominent in the lungs and spleen. No hybridization signal was detected in three carcinoma cell lines examined in parallel. Northern blot analysis of poly A+ RNA isolated from solid tumors revealed a tetranectin specific mRNA band. In situ hybridizations on tissue sections of colon carcinomas and normal colon tissues revealed a strong and distinct hybridization signal of stromal cells in colon carcinomas but not in tumor cells. Only a few stromal cells were labeled in the normal colon. Immunohistochemically, tetranectin was found in a fibrillar-like pattern in the extracellular matrix around the tumor islands and was not detectable in the normal colon stromal tissue. Plasminogen exhibited a similar immunohistochemical staining pattern as tetranectin. Human tetranectin cDNA comprises 874 base pairs including a 606-base pair open reading frame encoding 202 amino acids including a classical signal peptide. This protein is produced locally by cells of the stromal compartment of tumors and is deposited into the extracellular matrix. Since tetranectin binds to plasminogen we hypothesize that it could function as an anchor and/or reservoir for plasminogen and similar substances that regulate tumor invasion and metastasis as well as tumor angiogenesis.


Prevention of serious cardiac events by low-dose aspirin in patients with silent myocardial ischaemia. The Research Group on Instability in Coronary Artery Disease in Southeast Sweden. On exercise testing after an episode of unstable coronary artery disease (CAD; unstable angina or non-Q-wave myocardial infarction), a proportion of patients show ST-segment depression, indicating myocardial ischaemia, but do not report concomitant symptoms of angina. Treatment of such "silent" ischaemia aims mainly to reduce the risk of subsequent cardiac events. We have studied the effect of low-dose aspirin in patients with myocardial ischaemia defined at the predischarge test as silent (though patients might have had symptomatic ischaemia at other times) or symptomatic. 740 men with unstable CAD aged 70 years or less underwent symptom-limited exercise testing before hospital discharge; 144 showed ST depression without pain and 230 ST depression with simultaneous chest pain. Of the silent ischaemia group, 67 were randomly assigned placebo and 77 aspirin (75 mg daily); the corresponding numbers in the symptomatic group were 125 and 105. Angina symptoms were less common in the silent than in the symptomatic ischaemia group both before inclusion and during follow-up, and a greater proportion of the silent ischaemia group were included because of myocardial infarction. In both ischaemia groups aspirin treatment reduced the risk of subsequent myocardial infarction or death by 3 months' follow-up (silent 4% of aspirin-treated vs 21% of placebo-treated patients, p = 0.004; symptomatic 9% vs 18%, p = 0.05); at 12 months' follow-up a significant benefit of aspirin was still apparent in the silent ischaemia group (9% vs 28%, p = 0.005) but not in the symptomatic group (13% vs 22%, p = 0.109). Low-dose aspirin reduced the risk of subsequent myocardial infarction at least as well in silent as in symptomatic myocardial ischaemia. Since improvement of outlook is the main treatment objective in symptom-free patients, aspirin should be a mainstay of their treatment.


Randomised controlled trial of adjuvant chemotherapy by portal-vein perfusion after curative resection for colorectal adenocarcinoma. About half the patients treated with curative resection for colorectal cancer do not survive long-term. Adjuvant chemotherapy given during and after surgery may prevent hepatic metastases and improve patient survival. In patients with colorectal cancer, we have done a multicentre, randomised controlled trial comparing five-year survival after intraportal infusion of fluorouracil (1 g per day) plus heparin (10,000 U per day) (130 patients) or heparin alone (123) during curative resection and for 7 days thereafter, or after resection alone (145). There was no reduction in liver metastasis or increased overall survival advantage in either active-treatment arm of the study. However, patients who had stage III, Dukes' C (lymph-node-positive) tumours resected and were treated with fluorouracil plus heparin had a significant (p less than 0.03) survival advantage of about 16% compared with surgery-only controls. Further study of intraportal infusion of chemotherapeutic agent as adjuvant treatment to surgery in patients with colorectal cancer appears worthwhile.


The use of radioreceptor assays for the determination of benzodiazepines in biological samples: a review. Benzodiazepines are the most widely prescribed class of psychotropes. The demonstration of specific, high affinity binding sites for benzodiazepines in mammalian brain has provided a basis for a radioreceptor assay (RRA) of these compounds in biological samples (fluids or tissues). The RRA permits the simultaneous measurement of the benzodiazepine molecules that bind to the receptor, providing a total estimate of all pharmacologically active forms of the drug, which is useful in drug monitoring and in the intensive care of patients. After a complete description of the methodological aspects of this technique, the results obtained in therapeutic monitoring and in toxicological analysis are reviewed, and the advantages and disadvantages of this method are examined.


Effects of ribose on exercise-induced ischaemia in stable coronary artery disease. There is no established treatment specifically aimed at protecting or restoring cardiac energy metabolism, which is greatly impaired by ischaemia. Even after reperfusion, myocardial content of ATP remains low for more than 72 h. Long-term post-ischaemic dysfunction and irreversibility of ischaemic damage have been associated with low ATP content. Evidence that the pentose sugar ribose stimulates ATP synthesis and improves cardiac function led us to test the possibility that ribose increases tolerance to myocardial ischaemia in patients with coronary artery disease (CAD). 20 men with documented severe CAD underwent two symptom-limited treadmill exercise tests on 2 consecutive days; we postulated that the ischaemia induced might bring about changes in ATP metabolism lasting for several days. Patients whose baseline tests showed reproducibility were randomly allocated 3 days of treatment with placebo or ribose 60 g daily in four doses by mouth. Exercise testing was repeated after treatment on day 5. At that time mean (95% confidence interval) treadmill walking time until 1 mm ST-segment depression was significantly greater in the ribose than in the placebo group (276 [220-331] vs 223 [188-259] s; p = 0.002). The groups did not differ significantly in time to moderate angina. In the ribose-treated group the changes from baseline to day 5 in both time to ST depression and time to moderate angina were significant (p less than 0.005), but these changes were not significant in the placebo group. In patients with CAD, administration of ribose by mouth for 3 days improved the heart's tolerance to ischaemia. The presumed effects on cardiac energy metabolism offer new possibilities for adjunctive medical treatment of myocardial ischaemia.


Antibodies to silicone elastomers and reactions to ventriculoperitoneal shunts. Silicone elastomers used to make medical implants and prostheses are generally believed to be biologically inert. However, we have seen two patients who showed severe, apparently immunemediated, reactions to ventriculoperitoneal (VP) shunts. We used an enzyme-linked immunosorbent assay in which Silastic tubing served as the solid-phase antigen to test serum from the two patients, five other VP shunt patients without inflammatory reactions, and nine healthy adults. IgG binding to Silastic tubing was consistently higher in the two patients than in the healthy or patient controls. The IgG seemed to be binding specifically, since IgG Fab fragments also bound to the tubing, and preincubation of serum with Silastic or silylated proteins removed most of the activity. These findings show that specific immune reactivity to elastomers of polydimethylsiloxane can develop in human beings.


Cardiac resuscitation with percutaneous cardiopulmonary support. Percutaneous cardiopulmonary support (CPS) was initiated in 9 patients to provide haemodynamic stability after failure of conventional resuscitation. 4 patients were in cardiogenic shock and 4 remained in asystole, with 1 in resistant ventricular fibrillation, after cardiac arrest. During CPS for those in cardiogenic shock, the mean intra-arterial pressures ranged from 65 to 100 mm Hg (mean 84), at flow rates of between 3 to 5 l/min (mean 3.9). 2 patients underwent technically successful coronary angioplasty. No patient in this group survived. In the cardiac arrest group, acceptable mean intra-arterial blood pressures were achieved (mean 95, range 90-100 mm Hg) at flow rates of between 2 to 3 l/min (mean 2.6). All 5 subjects underwent technically successful coronary angioplasty whilst on CPS. 4 survived. 2 were alive and well at 12 months follow-up, 1 of whom had returned to work; the third is alive and well at 4 months.


Should patients with Björk-Shiley valves undergo prophylactic replacement? About 85,000 patients have undergone replacement of diseased heart valves with prosthetic Björk-Shiley convexo-concave (CC) valves. These valves are prone to fracture of the outlet strut, which leads to acute valve failure that is usually fatal. Should patients with these valves undergo prophylactic replacement to avoid fracture? The incidence of strut fracture varies between 0% and 1.5% per year, depending on valve opening angle (60 degrees or 70 degrees), diameter (less than 29 mm or greater than or equal to 29 mm), and location (aortic or mitral). Other factors include the patient's life expectancy and the expected morbidity and mortality associated with reoperation. We have used decision analysis to identify the patients most likely to benefit from prophylactic reoperation. The incidence of outlet strut fracture was estimated from the data of three large studies on CC valves, and stratified by opening angle, diameter, and location. A Markov decision analysis model was used to estimate life expectancy for patients undergoing prophylactic valve replacement and for those not undergoing reoperation. Prophylactic valve replacement does not benefit patients with CC valves that have low strut fracture risks (60 degrees aortic valves and less than 29 mm, 60 degrees mitral valves). For most patients with CC valves that have high strut fracture risks (greater than or equal to 29 mm, 70 degrees CC), prophylactic valve replacement increases life expectancy. However, elderly patients with such valves benefit from prophylactic reoperation only if the risk of operative mortality is low. Patient age and operative risk are most important in recommendations for patients with CC valves that have intermediate strut fracture risks (less than 29 mm, 70 degrees valves and greater than or equal to 29 mm, 60 degrees mitral valves). For all patients and their doctors facing the difficult decision on whether to replace CC valves, individual estimates of operative mortality risk that take account of both patient-specific and institution-specific factors are essential.


Effect of blood transfusion on survival among children in a Kenyan hospital. In Africa, blood transfusions are frequently given to treat severe paediatric anaemia. Because of the risk of HIV transmission, identification of when transfusion will reduce the risk of death for severely anaemic children has become increasingly important. For all children admitted to a Kenyan hospital from October, 1989, to October, 1990, we collected data on clinical presentation, haemoglobin (Hb), receipt of transfusion, and in-hospital survival. Of 2433 admissions, 29% (684) had severe anaemia (Hb less than 5.0 g/dl), and 20% (483) received blood transfusions. Based on laboratory criteria only, children with Hb less than 3.9 g/dl who were transfused had lower mortality than those with Hb less than 3.9 g/dl who were not transfused, but this finding applied only to children transfused on the day of admission (odds ratio [OR] 0.30; 95% Cl 0.14, 0.61) or the day after admission (OR 0.37; 95% Cl 0.14, 1.00). Based on a combination of laboratory and clinical criteria, children with clinical signs of respiratory distress and Hb less than 4.7 g/dl who were transfused had lower morality than those who were not (OR 0.19; 95% Cl 0.09, 0.41). Among children without respiratory distress, there was no association between receipt of transfusion and mortality, irrespective of admission Hb. The frequency of blood transfusion can be reduced and survival enhanced by targeting blood to those children with severe anaemia and clinical signs of respiratory distress, and by using transfusion early in the course of hospitalisation.


Meta-analysis of intervention trials on case-management of pneumonia in community settings. To appraise the effectiveness of the pneumonia case-management strategy in improving child survival, we have done a meta-analysis of six published intervention trials. The results of a seventh published study and two unpublished studies were also reviewed. The six published studies satisfied our criteria for methodological soundness. The reduction in mortality rate (control group minus intervention group) was estimated for each study, and for all the studies together. For total infant mortality, the overall reduction was 15.9 (95% confidence interval 10.6-21.1) deaths per 1000 livebirths; infant mortality due to acute lower respiratory infection was reduced by 10.7 (4.8-16.7) deaths/1000 livebirths. Mortality among children under 5 years was decreased by 36 deaths/1000 livebirths. The pooled estimates of relative risk are consistent with a 20% reduction in infant mortality and a 25% reduction in under-5 mortality. There was no clear association across the studies between the effect of the pneumonia case-management and extent of co-interventions such as immunisation and oral rehydration therapy. The consistency of findings of all the studies, despite differences in design and methods, shows that the case-management strategy has a substantial effect on infant and under-5 mortality, at least in settings with infant mortality rates of 90/1000 livebirths or more. It is important to find out the most efficient ways of implementing this strategy and integrating it into primary health care.


Requisite structural characteristics for benzodiazepine inhibition of triiodothyronine uptake into a human liver cell line. Previously, we reported potent inhibition of triiodo-L-thyronine (T3) cellular uptake into a human liver cell line (HepG2) by central and peripheral receptor specific benzodiazepine (BZ) compounds and our working hypothesis of BZ's as direct competitors for the iodothyronine transporter, displacing T3 but not acting as a substrate for transport. In this report, we list other reported uptake inhibitors and compare them to 23 benzodiazepine receptor ligands, in their potency to inhibit cellular uptake of T3. The most potent inhibitors are restricted to the benzodiazepine class. From the BZ structure-activity relationship (SAR) for inhibition, we see that the nonfused phenyl ring may be essential for activity and the strongest relationship is seen with substitution at R2' where Cl greater than F greater than H. Substitution at R4' and hydroxyl substitution at R3 enhances potency as will alkyl groups at R1 or on the imidazole group in the 1,2-annelated series. With R7 substitution, Cl is preferred over NO2 but not necessarily H when R4' = Cl; this may reflect a slightly different orientation of the molecule with large aliphatic R1 groups and/or R4' substitution. The carbonyl at R2 in the 1,4 benzodiazepine series, enhances their potency. The resultant structure-activity relationship highlights the importance of the halogen-substituted nonfused phenyl ring and seems unique relative to other described benzodiazepine sites and/or effects.


Adrenoceptor mechanisms in the cardiovascular effects of cocaine in conscious squirrel monkeys. The purpose of the current experiment was to study the role of various adrenoceptor subtypes in the cardiovascular response to cocaine in conscious squirrel monkeys. A variety of adrenoceptor antagonists were administered i.v. prior to the administration of 0.3 mg/kg cocaine (i.v.). Cocaine alone produced an increase in both blood pressure and heart rate. The non-selective alpha adrenoceptor antagonist phentolamine produced a dose-dependent antagonism of the pressor effect of cocaine, as did the alpha-1 selective antagonist prazosin. The alpha-2 selective antagonist yohimbine had no effect on the pressor effect of cocaine. The non-selective beta antagonist propranolol enhanced the pressor effect of cocaine as did the beta-1 selective antagonist atenolol. However, the effect of atenolol was not dose-dependent. The beta-2 selective antagonist ICI 118,551 and labetalol, which blocks both alpha and beta adrenoceptors, did not alter the pressor effect of cocaine. Propranolol, atenolol, and labetalol all antagonized the tachycardiac effect of cocaine in a dose-dependent manner, while the beta-2 antagonist ICI 118,551 did not. Phentolamine, prazosin and yohimbine also reduced the tachycardiac effect of cocaine, although these effects were dose-dependent only for yohimbine, which also significantly elevated baseline heart rate. These results indicate that alpha-1 adrenoceptor mechanisms mediate the pressor effect of cocaine, while beta-1 adrenoceptor mechanisms are involved in the tachycardiac effect of cocaine in squirrel monkeys. Propranolol potentiated cocaine's pressor effect through beta-2 independent mechanisms. Thus, neither alpha-2 nor beta-2 adrenoceptor mechanisms appear to be involved in cocaine's cardiovascular effects.


Short echo time proton spectroscopy of human brain using a gradient head coil. Short echo time, single voxel localized proton spectroscopy was accomplished using a stimulated echo (STEAM) sequence running on a Siemens 1.5-T system with a head coil incorporating the Z and Y gradients. Spectra from the temporal lobe, the cerebellum and mid brain were acquired from a group of normal volunteers using the following parameters: voxel size = 8 ml, TE = 22 msec, 512 signal averages and TR = 1.7 sec. STEAM spectra acquired with the small diameter gradients showed significantly fewer artifacts at short TE, allowing the observation of glutamate/glutamine, GABA, taurine, and inositol in addition to the prominent resonance of choline, creatine/phosphocreatine and N-acetylaspartate (NAA). The levels of chlorine, creatine and NAA were found to be significantly different in the three regions of the brain examined.


Age and serotype dependent binding of K88 fimbriae to porcine intestinal receptors. The porcine small intestine contains several polypeptides that could function as receptors for K88-positive Escherichia coli. The mucus fraction contained three proteins with molecular weights of 25, 35 and 60 kDa respectively, which showed a high affinity for K88-positive E. coli cells, whereas brush borders contained a 16 kDa protein and a set of proteins ranging from 40-70 kDa. Depending on the K88 serotype tested, differences in binding to these proteins were observed. In particular, E. coli cells carrying K88ad fimbriae exhibited only a rather weak binding to mucus proteins. The influence of age of the pig on the presence of K88 receptors was also investigated. One-week-old and 35-days-old post-weaning piglets were shown to contain K88 receptors in their mucus while these receptors were hardly detectable in the mucus of 6-month-old pigs. The presence of receptors in the brush border fraction was shown to be independent of age. The binding of K88 fimbriae to mucus proteins was blocked using a lectin of Euonymus europeaus which specifically recognizes the Gal alpha(1-3)Gal sequence, indicating that this disaccharide forms a significant part of the receptor structure.


GM-CSF production by CD4+ T-lymphocytes is selectively impaired during the course of HIV-1 infection. A possible indication of a preferential lesion of a specific subset of peripheral blood CD4+ T-lymphocytes. The production of granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) were evaluated in the supernatants of short-term cultures of purified CD4+ T-lymphocytes and enriched monocytes obtained from peripheral blood (PB) of 35 HIV-1 seropositive (+) asymptomatic individuals, stages I-II of the Walter Reed (WR) classification, 15 HIV (+) symptomatic patients (WR V-VI) and 40 HIV-1 seronegative normal blood donors. IL-1 beta and TNF-alpha production by either enriched monocytes or isolated CD4+ T-cells, was similar in HIV-1 (+) asymptomatic, symptomatic subjects and normal controls. GM-CSF level in enriched monocyte culture supernatants did not show any significant difference in the three groups of subjects under investigation. On the other hand, GM-CSF production by isolated CD4+ T-lymphocytes was two-fold decreased in HIV-1 (+) asymptomatic subjects and five-fold decreased in HIV-1 (+) symptomatic patients with respect to normal blood donors. The decline in GM-CSF production was clearly correlated with viral isolation from patient's PB light density mononuclear cells (r = -0.920, p less than 0.01). The selective and progressive decline in GM-CSF production by CD4+ T-lymphocytes, starting from early stages of HIV-1 infection, suggest a preferential lesion of a specific subset of CD4+ T-lymphocytes characterized by an intense production of GM-CSF and may contribute to explain the deranged inflammatory and immune responses which characterize the course of HIV-1 infection.


Application of immunohistochemical techniques to sural nerve biopsies. The role of immunohistochemistry in the day-to-day diagnostic work of a peripheral nerve laboratory is not yet clearly established, although for conditions such as amyloid neuropathy, immunohistochemistry appears to be a useful adjunct to conventional techniques. Immunohistochemistry has provided new information about some neuropathies in which immune dysfunction is believed to play a central role. Immunohistochemical data about normal human nerve are scarce; a better appreciation of the normal cellular constituents of nerve, particularly the endoneurium, is needed. In the future, the techniques may be a means to understand better the pathogenesis of other types of neuropathy, such as inherited or toxic neuropathies, or to examine fundamental pathologic events such as axonal degeneration.


Availability of flow cytometric immunophenotyping of lymphocytes to hospital patients--United States, 1990. The pathogenesis of disease caused by human immunodeficiency virus (HIV) is largely attributable to the decrease in T-lymphocytes bearing the CD4 cell-surface molecule (CD4 + T-lymphocytes) (1). The percentage of CD4 + T-lymphocytes among total lymphocytes and the percentages of other lymphocyte subpopulations (e.g., CD8 + T-lymphocytes) are generally measured by flow cytometric immunophenotyping (FCl) (also called immunophenotyping by flow cytometry [2], T-lymphocyte immunophenotyping [3], and fluorescence-activated cell sorting). FCl results are frequently used to guide the treatment of HIV-infected persons. To assess the availability of FCl to hospital patients, in 1990, the National Public Health and Hospital Institute (NPHHI), a private, nonprofit research institute, surveyed hospitals about their provision of FCl to patients. This report presents findings from the survey.


Identification of a novel transglutaminase from the filarial parasite Brugia malayi and its role in growth and development. Recently, we reported the presence of a putative transglutaminase in adult female worms of Brugia malayi [1]. The enzyme activity was shown to be essential for in utero growth and development of microfilariae. Here, we demonstrate that adult worms of B. malayi have a large amount of epsilon-(gamma-glutamyl)lysine isopeptide bonds, a product of physiologically active transglutaminase. A 25-kDa immunoreactive band detected in female worm extracts by a monospecific monoclonal antibody (CUB 7401) against guinea pig liver transglutaminase was associated with the enzymatic activity. Unlike the mammalian enzyme, the parasite enzyme did not require Ca2+ for its catalytic activity. Furthermore, in utero developing embryos, especially during early stages of development, contained very high amounts of this enzyme. Adult female worms contained several proteins that could serve as suitable substrates for the enzyme. Inhibition of the enzyme activity by an enzyme-specific pseudosubstrate, monodansylcadaverine, led to a time- and dose-dependent inhibition of microfilariae production and release by gravid female worms. The inhibition of microfilariae production was due to the inhibition of transglutaminase-catalyzed crosslinking of parasite proteins that in turn seemed to be essential for in utero growth and development of the embryos. The results suggest that transglutaminase-catalyzed reactions may play an important role during early development of embryos to mature microfilariae inside the adult female worms of filarial parasites.


[The effect of 2,4-dichlorophenol on enzyme activities and other parameters in parenchymatous organs of rats]. After repeated administration of 2,4-dichlorophenol the aminopeptidases AAP and LAP, and the alkaline phosphatase in various organs were influenced. The treatment causes stimulatory or inhibitory activities. Also with histological and histochemical methods damages could be detected.