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At least 19 recordsLinked to original sources
PubMed1991

Heparin sensitivity and resistance in the neonate: an explanation.

Based on in vitro tests, newborns are reported to be both sensitive and resistant to standard heparin (SH) compared to adults. The sensitivity to SH occurs in assays based on de novo thrombin generation, and SH resistance occurs in systems where exogenous thrombin is added to newborn plasma. We hypothesized that this apparent paradox is related to the antithrombin III to (pro) thrombin ratio associated with each test. Since SH catalyses the activity of antithrombin III, any imbalance in the antithrombin III/(pro)thrombin ratio in newborns compared to adults would be amplified by SH. If the ratio of antithrombin III/(pro)thrombin is defined as 1 in adults, in comparison newborns have a ratio of 1.5. We compared how various doses of SH (0.1 to 0.6 u/ml) inhibited the generation of endogenous thrombin in defibrinated newborn (N) and adult (A) plasma. Following contact activation and recalcification of each plasma, thrombin activity was measured using a chromogenic substrate and quantitated by measuring the area under the curve. In the presence of SH, newborn plasma was more sensitive to SH than adult plasma and generated relatively less thrombin (N:6.1 vs A:9.1% sec/u/ml; p less than 0.01). When the ratio of antithrombin III/(pro)thrombin in newborns was altered to 2.5 by exogenous antithrombin III, the SH sensitivity was increased. This plasma now generated no detectable thrombin in the presence of only 0.1 u/ml of SH. In contrast, when the ratio of antithrombin III/(pro) thrombin of the newborn was altered by exogenous prothrombin to 0.6, this plasma now became resistant to SH and generated more thrombin than adults in the presence of SH (N:11.6 vs A:9.1; p less than 0.01). Because of the potential use of low molecular weight heparins (LMWH) in newborns, a LMWH, Choay 222, was also tested in a similar fashion and gave similar results to SH. Thus, the ratio of antithrombin III/(pro)thrombin likely determines the in vitro sensitivity or resistance of newborn plasma to SH and LMWH. There are no clinical studies that determine if newborns require more or less SH than the adult to successfully treat thrombotic complications. Current practice is a simple extrapolation of therapeutic ranges from adults. We speculate that the efficacy and safety of SH in the newborn in vivo can be improved by altering the antithrombin III/(pro)thrombin ratio.

Adult↗
PubMed1996

Transgenic mice expressing the Sh ble bleomycin resistance gene are protected against bleomycin-induced pulmonary fibrosis.

Despite the high efficiency of bleomycin (BLM) as a chemotherapeutic agent against various carcinomas, the potentially lethal and chronic fibrotic response of the lung is a major dose-limiting side effect. Here, we explore the possibility of a direct inhibition of lung tissue injury by in vivo expression of the actinomycetes BLM resistance protein Sh ble. Transgenic mice expressing the Sh ble gene under the control of a composite viral promoter were produced after introduction of the transgene into D3 ES cells. The protein was detected at high level in lungs, spleen, and kidney. We then assessed its ability to modulate the BLM-induced fibrotic response in the transgenic mice in comparison with C57BL/6 and 129/Sv parental mice. Cumulative doses of 300, 400, or 500 mg/kg BLM were administered either by i.p. or s.c. repeated injections in the different strains. Transgenic mice were shown to be clearly less sensitive to BLM toxicity, as assessed by lung histology. The pulmonary hydroxyproline content in the treated transgenic mice was close to its baseline level, whereas it was up to 50% higher than the control level in C57BL/6 and 129/Sv parental mice. These observations are consistent with the hypothesis that a resistance gene specifically expressed in lungs may prevent the BLM-induced inflammation.

Acetyltransferases↗
PubMed2026

Integrated Functional Characterization of Hemileia vastatrix Effector Candidates Reveals Coordinated Immune Suppression, Sequential Deployment and Compartment-Specific Targeting.

Coffee leaf rust, caused by the obligate biotrophic fungus Hemileia vastatrix, remains the most destructive disease of coffee worldwide. Although genomic and transcriptomic studies have identified a large number of candidate effectors, experimental evidence supporting their biological roles during infection remains limited. Here, we integrated functional assays, temporal expression profiling during coffee infection and subcellular localization analyses to investigate the biological properties of 44 H. vastatrix effector candidates (HvECs). Using the Pseudomonas fluorescens EtHAn effector delivery system in Nicotiana benthamiana, 15 HvECs consistently suppressed pattern-triggered immunity (PTI), indicating that immune suppression is a widespread property among the H. vastatrix effector repertoire, as assessed in this heterologous system. Five HvECs also attenuated AvrB-triggered effector-triggered immunity (ETI), and three suppressed both PTI and ETI, suggesting that a subset of HvECs targets conserved regulatory nodes shared by these interconnected immune pathways. Temporal expression profiling revealed sequential deployment of HvECs throughout infection, with distinct subsets predominating during pre-biotrophic development, host penetration or biotrophic colonization, consistent with stage-specific functions during fungal pathogenesis. Subcellular localization analyses further showed that HvECs preferentially accumulated in the nucleus or chloroplasts, compartments known as central hubs of plant immune regulation. This study provides the most comprehensive functional characterization of H. vastatrix effector candidates to date, establishes a biologically informed framework for prioritizing candidates for future identification of avirulence determinants recognized by SH resistance genes, and advances our understanding of how the coffee rust fungus orchestrates immune suppression across time and cellular space during pathogenesis.

Nicotiana↗
PubMed1996

Generation of small fusion genes carrying phleomycin resistance and Drosophila alcohol dehydrogenase reporter properties: their application in retroviral vectors.

We have used the Drosophila ADH cDNA to engineer new fusion genes carrying both reporter activity and bleomycin/phleomycin resistance (Sh ble). Cassettes of ADH::Sh ble, Sh ble::ADH, or ADH::Sh ble::ADH with or without polyadenylation signals were constructed. Placed under the control of the strong CMV promoter, these constructs induced intense ADH substrate staining and phleomycin resistance, whatever the position of the ADH gene, in avian or mammalian cell lines. SW-based nonreplicative retroviral vectors were constructed and introduced into the appropriate packaging cell line. Titers up to 10(6) ADH forming units/ml of viral supernatant were obtained except for the ADH::Sh ble::ADH construct, which reached 10(5) ADH forming units. These retroviral vectors were inoculated to the E3 chick embryo via the coelom. Three days later, cells from different organs were put in culture for 24 h and stained to detect ADH activity. A large number of positive cells were found in cultures from all organs. The new fusion genes described here are, to our knowledge, the smallest (1.1 kb) published to date that carry both reporter and drug resistance properties. These genes represent the basis of a new retroviral vector model with three distinct properties in two genetic units; their advantage is to reduce the size and increase the efficiency of the vector.

Alcohol Dehydrogenase↗
PubMed2000

Comparative evaluation of different rat models with co-existing diabetes-mellitus and hypertension.

We have evaluated the suitability of different rat models for the study of effects of antihypertensives on cardiovascular and metabolic complications of diabetes mellitus and hypertension. IDDM was induced in Wistar and spontaneously hypertensive (SH) rats by single tail vein injection of STZ (45 mg/kg, i.v.). Neonatal STZ-diabetes (nSTZ) was induced by administering STZ, 70 mg/kg (i.p.) to 5 day old Wistar rat pups. DOCA-hypertension was induced in Wistar and STZ-diabetic rats using deoxycorticosterone acetate (DOCA, 5 mg/kg, s.c.) and NaCl (2%) in drinking water. Intravenous injection of STZ produced cardinal signs of diabetes mellitus including hyperglycemia, loss of body weight, polyphagia and polydipsia. STZ-diabetic rats also showed hyperlipidemia and hypoinsulinemia. STZ-treated rats developed hypertension and bradycardia. nSTZ rats were found to have mild hyperglycemia and were hypertensive and hyperinsulinemic. The OGTT and ITT revealed that nSTZ rats are insulin resistant. SH rats were also found to be hyperinsulinemic and hypertensive. Although, these rats were found to be insulin resistant, they did not demonstrate hyperglycemia. DOCA-treated STZ-diabetic rats were found to have milder hyperglycemia when compared to STZ-diabetic rats not treated with DOCA. Although, DOCA treatment was not found to alter serum levels of glucose and insulin, results of OGTT revealed enhanced glucose disposal in DOCA-treated Wistar rats, suggesting that DOCA probably produces some effect on glucose homeostasis in rats. The present data also suggest that STZ-diabetic rat may be considered a suitable model for IDDM. On the other hand, nSTZ and SH rats were hyperinsulinemic and insulin resistant and may be used as models to study insulin sensitivity. DOCA-hypertensive rat may not be a suitable model for studying the effects of various drug interventions on glucose homeostasis and insulin sensitivity as DOCA itself appears to influence these factors.

Animals↗
PubMed1991

Proteolytic events in the processing of secreted proteins in fungi.

Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.

Amino Acid Sequence↗
PubMed2002

Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea.

Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 microg of fGH protein expression per one liter culture containing 1 x 10(8) cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding.

Journal Article↗
PubMed1992

Efficient secretion of human lysozyme fused to the Sh ble phleomycin resistance protein by the fungus Tolypocladium geodes.

Tolypocladium geodes strain NC50 was transformed by different integrating vectors bearing both a synthetic gene encoding human lysozyme (HLz) and the Sh ble phleomycin resistance marker, either in separate expression cassettes or in transcriptional or translational fusion configurations. Clones derived from all vectors were able to secrete HLz. The highest productivities in shake flasks (up to 150 mg l-1 in 5 days) were obtained when HLz was fused at the C-terminal end of the Sh ble protein. The fusion protein is efficiently secreted and release of active lysozyme occurs by extracellular proteolytic cleavage in the junction peptide.

Amino Acid Sequence↗
PubMed1980

[Characteristics of attenuated immunogenic mutants of shigella flexneri selected from phosphomycin-resistant clones].

Sh. flexneri mutants No. 2a, resistant to phosphomycin (25 microgram/ml), differ in their fermentability in respect to glucose, mannitol, mannose and fructose. The mutants fall into 7 groups by this characteristic. All the resistant mutants, irrespective of the type of hydrocarbon fermentation, have lost their ability of causing keratoconjunctivitis. The decreased virulence of the phosphomycin-resistant mutants is linked with the loss of their penetration properties, although their adhesiveness and proliferative capacity on the surface HeLa cells is retained. A change in the virulence of the mutants under study is not connected with disturbances in the synthesis of O-antigen LPS. Most of these mutants are highly immunogenic.

Animals↗
PubMedDate not supplied

Enhanced expression of the multidrug resistance gene in vindesine-resistant human esophageal cancer cells.

We developed and characterized a new series of low- and high-grade multi-drug-resistant (MDR) cell lines of human esophageal carcinoma. Eight vindesine-resistant clones, SH-1-V1, SH-1-V2, SH-1-V3, SH-1-V4, SH-1-V5, SH-1-V6, SH-1-V7, and SH-1-V8 were isolated from the human esophageal cancer cell line, SH-1, by stepwise selection on exposure to increasing doses of vindesine. SH-1-V1 to SH-1-V8 acquired resistance to vindesine, in a stepwise manner, from 3- to 115-fold over findings in the parental SH-1 cells. The most resistant clone, SH-1-V8, was cross-resistant to other anticancer agents such as vincristine, actinomycin D, and daunomycin, thereby suggesting acquisition of the MDR phenotype. In SH-1-V8 cells, cellular accumulation of vincristine decreased and an MDR reversal agent, cepharanthine, potentiated the cytocidal action of vindesine. The expression of MDR1 mRNA was enhanced and amplification of the MDR1 gene was observed in clones SH-1-V4, SH-1-V5, SH-1-V6, SH-1-V7 and SH-1-V8; expression of MDR1 mRNA was detectable without gene amplification in the remaining 3 clones. The enhanced expression of the MDR1 gene may be involved in the acquisition of vindesine resistance in human esophageal cancer cells.

Alkaloids↗
PubMed2000

Voltage clamping single cells in intact malpighian tubules of mosquitoes.

Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage (V(pc)) was -86.7 mV, and input resistance (R(pc)) was 388.5 kOmega (n = 49 cells). In six cells, Ba(2+) (15 mM) had negligible effects on V(pc), but it increased R(pc) from 325.3 to 684.5 kOmega (P < 0.001). In the presence of Ba(2+), leucokinin-VIII (1 microM) increased V(pc) to -101.8 mV (P < 0.001) and reduced R(pc) to 340.2 kOmega (P < 0.002). Circuit analysis yields the following: basolateral membrane resistance, 652. 0 kOmega; apical membrane resistance, 340.2 kOmega; shunt resistance (R(sh)), 344.3 kOmega; transcellular resistance, 992.2 kOmega. The fractional resistance of the apical membrane (0.35) and the ratio of transcellular resistance and R(sh) (3.53) agree closely with values obtained by cable analysis in isolated perfused tubules and confirm the usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized V(pc) from -94.3 to -10.7 mV (P < 0.001) and reversibly increased R(pc) from 412 to 2,879 kOmega (P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways.

Animals↗
PubMed1981

Increase in drug resistance among Shigella dysenteriae, Sh flexneri, and Sh boydii.

Two thousand three hundred and seventy strains of Shigella dysenteriae, Sh flexneri, and Sh boydii isolated in England and Wales from 1974 to 1978 were tested for resistance to 12 antimicrobial drugs. Eighty per cent of strains were resistant to one or more drugs, with sulphonamide resistance occurring most frequently. Resistance to streptomycin, tetracycline, ampicillin, and chloramphenicol increased during the period, as did the incidence of multiple resistance. Most infections due to Sh dysenteriae, Sh flexneri, and Sh boydii are acquired abroad, and the increasing incidence of drug resistance among these organisms contrasts with the decreasing incidence of resistance among the indigenous Sh sonnei. These findings may indicate the need for better control of antibiotic use, particularly in developing countries.

Anti-Bacterial Agents↗
PubMed1997

Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis.

Acetyltransferases↗