Acute effects of various doses of iodide on thyroid iodine autoregulation

Analytical ion microscopy (AIM) was used to determine alterations in the thyroid follicular lumen¹²⁷I stores of Wistar rats injected with different doses of¹²⁹I (low specific activity radionuclide). Animals fed a normal iodine diet (10 μg¹²⁷I/d) were divided into four groups: control group and three treated groups injected ip 24 h before sacrifice with¹²⁹I at doses of 10 μg (group 1), 30 μg (group 2), and 8500 μg (group 3). AIM was performed on thyroid semithin sections. The mean¹²⁹I concentration increased with the dose injected from group 1 (0.44±0.03 μg/mg, mean ± SEM) to group 2 (2.05±0.14 μg/mg) and decreased in group 3 (0.57±0.08 μg/mg). When compared to control group (4.14±0.17 μg/mg), the mean¹²⁷I concentration was not changed in group 1 (4.52±0.07 μg/mg), but altered in the other groups: It was significantly increased (7.14±0.41 μg/mg) in group 2 and slightly decreased (3.11±0.26 μg/mg) in group 3. These results underline the interest of AIM in the study of the effects of various doses of iodide on the thyroid autoregulation by iodide, a trace element actively trapped by this gland.     

Plant regeneration from protocorm-derived callus of <Emphasis Type="Italic">Cypripedium formosanum</Emphasis>

Summary Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the medium with 4.44 μM N⁶-benzyladenine after 8 wk of culture. The regenerated protocorm-like bodies formed shoots and roots on medium containing 1 g l⁻¹ activated charcoal and 20 g l⁻¹ potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established.     

Fungicidal effect of hydrogen peroxide on fungal infection of rainbow trout eggs

Exposure to 1,500 μg/ml of hydrogen peroxide (H₂O₂) for 60 min at 13°C was found to be injurious to rainbow trout eggs. On the other hand, the concentration which effectively inhibited pathogenic fungi in vitro was substantially less than this toxic dosage; specifically, 500 μg/ml for 60 min at 20°C to inhibit the zoosporic stage and 1,000 μg/ml for 60 min at 20°C to inhibit the vegetative stage. From in vivo tests, treatment with 1,000 μg/ml of H₂O₂ for 60 min at 13°C was found to be the most effective procedure to control fungal infection and increase the hatching rate of rainbow trout eggs.     

Direct effects of serotonin and ketanserin on the functional morphology of embryonic chick skin in vitro

Summary Different organotypical culture methods are used to test the direct effects of serotonin and ketanserin, a S₂, α₁, and H₁ receptor antagonist in vascular tissue, on fibroblasts and epidermal cells of embryonic chick skin in vitro. From light microscopic and electron microscopic analyses, we learn that serotonin enhances keratinization and differentiation, whereas ketanserin reduces differentiation in comparison to the control cultures. Incorporation data of fragments cultured with [³H]thymidine show that ketanserin, within a dose range from 0.05 to 5 μg/ml, stimulates proliferation. Serotonin at a concentration of 10 μg/ml slightly slows down proliferation, whereas lower doses of 0.1 and 1 μg/ml result in tritium activities that do not differ from control cultures. This investigation was financially supported by the National Fund of Scientific Research, Belgium, 3.0022.87.     

The effects of anhydrous ammonia on membrane stability ofP.hymatotrichum omnivorum

Sclerotia and mycelium ofPhymatotrichum omnivorum were exposed to anhydrous ammonia (NH₃) and then observed with an electron microscope in order to determine the effects of the NH₃ treatment on the fungal membranes. Sclerotia were exposed to four rates of NH₃: 28, 56, 84, and 112μg NH₃/ml of air for 24 hours. At 28μg/ml, the plasmalemma became wavy and the mitochondrial cristae began to swell and disperse. At 56μg NH₃/ml the plasmalemma showed breakage and formation of vesicles, and all other membrane systems within the cell were broken and distorted. All membranes were totally disrupted and no organelles were recognizable at 84μg NH₃/ml. Mycelium was exposed to 2, 4, 8, 20, and 40μg NH₃/ml for one minute. Damage to cell membranes was not observed at NH₃ conc. up to 4μg/ ml. At 8μg NH₃/ml the plasmalemma was broken and the mitochondria were disrupted. At 20μg/ ml and above, all internal organization was destroyed.     

Yersinia intermedia: A new species of enterobacteriaceae composed of rhamnose-positive,melibiose-positive,raffinose-positive strains (formerly calledYersinia enterocolitica orYersinia enterocolitica-like)

The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate (50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency, when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR⁺, HCR⁻, and irradiated HCR⁻ strains. These drugs increased mutation frequency and lethality of irradiated HCR⁺ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine. Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR⁺ cells. Our findings suggest that these chemicals selectively interfere with excision-repair.     

Fluconazole-, Amphotericin-B-, Caspofungin-, and Anidulafungin-Resistant <Emphasis Type="Italic">Candida ciferrii</Emphasis>: An Unknown Cause of Systemic Mycosis in a Child

Candida ciferrii, which is known as an agent of superficial yeast infection and onychomycosis, has rarely been isolated as an agent of candidemia. Limited reports have suggested different patterns of antifungal sensitivity. We report a rare candidemia case caused by c.ciferrii in an 8-year-old child in which isolated candida species were resistant to amphotericin-B (MIC > 1 μg/ml), fluconazole, (MIC ≥ 64 μg/ml), caspofungin (MIC ≥ 32 μg/ml), and anidulafungin (MIC ≥ 32 μg/ml) but sensitive to voriconazole (MIC ≤ 0.12 μg/ml). As far as we aware, this was the first recorded C. ciferrii candidemia case in children.     

Wound healing by a 3.2 kDa recombinant polypeptide from velvet antler of Cervus nippon Temminck

Velvet antler (VA) is used in traditional Chinese medicine to treat a wide range of ailments including the enhancement of wound healing. A 3.2 kDa recombinant polypeptide of VA from sika deer was purified and compared to native polypeptides stimulation growth of NIH3T3 cells. Both stimulated growth in a dose-dependent manner (10–100 μg/ml). To study its wound healing properties, burn-wounded rats were topically administered with recombinant VA polypeptide or native polypeptide. Rats treated with 0.05 and 0.1% (w/w) polypeptides exhibited significant wound healing. As the yield of recombinant polypeptide was 40-fold higher than that of the native polypeptide, it may therefore be a useful biopharmaceutical.     

Relative resistance to methothexate by proliferating normal rabbit epidermal cells in vitro

Summary Primary cell cultures of normal rabbit epidermal cells (keratinocytes) were established without the use of enzymatic techniques. Six experiments were carried out on cells from six different rabbits. When these cells were exposed to methotrexate (MTX) for 24 h at 1 μg/ml, proliferation, as measured by cells entering mitosis, was significantly inhibited (P<0.05) in only one experiment. When the dose of MTX was elevated to 100 μg/ml, only two experiments showed significant inhibition of mitosis. This minimal inhibition of mitosis by MTX was contrasted by the dramatic inhibitory effect of this antimetabolite on DNA synthesis. At 1 μg/ml MTX for 24 h, DNA synthesis, as measured by [³H]deoxyuridine uptake, was inhibited >95%. We can conclude that under certain conditions, the rabbit keratinocyte may represent a normal cell type that is inherently resistant to the antiproliferative effects of methotrexate. The research was supported by National Cancer Institute Grant CA 11536.     

Time-dependent expression of CD25 in phytohemagglutinin- or interleukin-2-stimulated human peripheral blood lymphocytes

The dynamics of the expression of the high-affinity receptor for interleukin-2 (IL-2 receptor) evaluated by the method of flow cytofluorimetry based on changes in the number of cells that express the CD25 marker (CD25+) was studied in human peripheral blood lymphocytes stimulated by various mitogens. It has been shown that, in the resting lymphocyte culture, both phytohemagglutinin (PHA, 10 μg/ml) and 12,13-phorbol dibutyrate (PDBu, 10⁻⁸ M) with ionomycin (IM, 5 × 10⁻⁷ M) induce a long-lasting increase (for 48 h) in the number of CD25+ cells. Interleukin-2 (IL-2) has only been found to be capable of inducing time-dependent CD25 expression in competent (not resting) lymphocytes pretreated with submitogenic doses of PHA (1 μg/ml). A comparison of the dynamics of the number of CD25+ cells and blast transformation has shown that CD25 markers are revealed as early as on small stimulated lymphocytes, while, at the late activation stages, which correspond to the stage of cell growth and transition to DNA synthesis, the overwhelming majority of blasts are CD25+ cells with high-affinity α -receptors for IL-2. The obtained data allow one to suggest that the expression of an α -subunit of IL-2 receptor takes place at the IL-2-dependent stage of T lymphocyte proliferation and may be directly induced by IL-2 via IL-2 receptor.     

Concentration-dependent differntial effects of cortisol on synthesis of α-lactalbumin and of casein in cultured mouse mammary gland explants: Importance of prolactin concentration

Summary Cortisol was previously shown to elicit a concentration-dependent inhibition of α-lactalbumin accumulation in midpregnant mouse mammary gland cultured in medium containing optimal concentrations of 5 μg/ml prolactin and insulin. In contrast, casein accumulation under these conditions was progressively stimulated by addition of increasing amounts of cortisol (Ono, M.; Oka, T. Cell 19: 473–480; 1980). In the present study we found that in the presence of a suboptimal concentration of 0.5 μg/ml prolactin, 2.8×10⁻⁹ M to 2.8×10⁻⁷ M cortisol stimulated α-lactalbumin accumulation. Furthermore, higher concentrations of cortisol produced a smaller inhibition of α-lactalbumin accumulation as compared to that obtained in cultures containing 5 μg/ml prolactin. The maximal increase in α-lactalbumin accumulation attained in the presence of 1.4×10⁻⁸ M cortisol, 0.5 μg/ml prolactin, and insulin was comparable to that observed in culture containing 5 μg/ml prolactin and insulin. Similar results were obtained in a cortisol concentration-response study of α-lactalbumin accumulation in cultures containing a suboptimal concentration of 0.5 μg/ml human placental lactogen. Measurement of the rate of α-lactalbumin synthesis in cultured tissue indicated that the opposing effects of low and high concentrations of cortisol on α-lactalbumin accumulation involved an alteration in the rate of synthesis of the milk protein. In contrast to α-lactalbumin, the synthesis of casein was stimulated in a concentration-dependent manner by addition of cortisol that acted synergistically with either 0.5 μg/ml or 5 μg/ml prolactin. The maximal increases were obtained in the presence of 2.8×10⁻⁶ M cortisol. These results indicated that the action of cortisol on α-lactalbumin accumulation can be modulated by the concentration, of prolactin and suggest that the interplay between cortisol and prolactin in regulation of α-lactalbumin synthesis may be different from that involved in casein synthesis.     

Induction of Haploid Morphogenic Calluses from in vitro Cultured Anthers of Prunus Armeniaca cv. ‘Harcot’

Apricot (Prunus armeniaca) ‘Harcot’ anthers, were cultured in vitro for the production of haploid plants. The best androgenic response was achieved with Nitsch and Nitsch (1969) medium, supplemented with 4.52 μM 2,4-D, 4.52 μM zeatin, 2.85 μM IAA and 40 g l⁻¹ sucrose. Cultures were maintained in the dark for 8 days, at 28°C, followed by transfer to a 16-h photoperiod, with 35 μm m⁻² s⁻¹ light intensity and 24/22°C day/night temperature. The androgenic response was correlated with the floral bud size, its phenologic stage and the level of microspore evolution. Anthers containing microspores at the tetrad/uninucleate stage were the most appropriate. The ploidy level of the calluses was evaluated by flow cytometry revealing that they range from haploid to octaploid. Mixoploid calluses have also been identified. Histological studies showed that the haploid calluses have their origin in the microspores. Nodular structures consisting of cells with dense cytoplasm and differentiated xylem elements were observed and were surrounded by an autofluorescent layer, probably due to cutin deposition.     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin (4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400 μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties. Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997     

Antiviral Activities of Marine Pseudomonas Polysaccharides and Their Oversulfated Derivatives

A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide. Among the antiviral activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration is 1.4 μg/ml). Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration is 11.0 μg/ml; for another, 2.9 μg/ml). Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1. No cytotoxicity of these products was noted against host cells at the 50% cytotoxic concentration of 100 μg/ml, except that naturally occurring sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8–21 μg/ml. Received May 1, 1998; accepted July 24, 1998.     

Study of the Action of Se and Cu on the Growth Metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> by Microcalorimetry

The biological effect of Se and Cu²⁺ on Escherichia coli (E. coli) growth was studied by using a 3114/3236 TAM Air Isothermal Calorimeter, ampoule method, at 37°C. From the thermogenesis curves, the thermokinetic equations were established under different conditions. The kinetics showed that a low concentration of Se (1–10 μg/mL) promoted the growth of E. coli, and a high concentration of Se (>10 μg/mL) inhibited the growth, but the Cu²⁺ was always inhibiting the growth of E. coli. Moreover, there was an antagonistic or positive synergistic effect of Se and Cu²⁺ on E. coli in the different culture medium when Se was 1–10 μg/ml and Cu²⁺ was 1–20 μg/ml. There was a negative synergistic effect of Se and Cu²⁺ on E. coli when Se was higher than 10 μg/ml and Cu²⁺ was higher than 20 μg/ml. The antagonistic or synergistic effect between Se and Cu²⁺ on E. coli was related to the formation of Cu–Se complexes under the different experimental conditions chosen.     

Effect of dikegulac on growth and alkaloid production inCatharanthus roseus (L.) G. Don. (pink flowered)

Application of sodium-dikegulac reduced plant height with associated increase in branch and leaf number and root biomass inC. roseus (L.) G. DON. Chlorophyll content reduced significantly after first month of 100 and 250 μg/ml DK application. However, such reduction was replaced by significant rise after forth month in 250 μg/ml DK application and fifth month in 100 μg/ml DK application followed by appreciable decline only in 250 μg/ml DK treatment but 100 μg/ml DK maintained higher level till harvest. Total sugar content was significantly high during forth and fifth month stage of growth after DK application. Amino acid content was higher during third to fifth month in 100 μg/ml DK treatment and during third to forth month in 250 μg/ml DK treatment. Tryptophan, on the other hand showed higher content at the fifth month stage of growth after application of DK in both the concentrations. Leaf and root dry weight as well as total alkaloid content were highest in 100 μg/ml DK application. DK, therefore, appears to be a potential chemical for increasing biomass and alkaloid content inC. roseus.     

Heavy metal resistant <Emphasis Type="Italic">Distigma proteus</Emphasis> (Euglenophyta) isolated from industrial effluents and its possible role in bioremediation of contaminated wastewaters

Summary The alga, Distigma proteus, isolated from industrial wastewater showed tolerance against Cd²⁺ (8.0 μg/ml), Cr⁶⁺ (12 μg/ml), Pb²⁺ (15 μg/ml) and Cu²⁺ (10 μg/ml). The metal ions slowed down the growth of the organism after 4–5 days of exposure. The reduction in cell population was 90% for Cu²⁺, 84% for Cd²⁺, 71% for Cr⁶⁺, and 63% for Pb²⁺ after 8 days of metal stress. The order of resistance to heavy metal, in terms of reduction in the cellular population, was Cu²⁺ > Cd²⁺ > Cr⁶⁺ > Pb²⁺. Chromium- and cadmium-processing capabilities of the alga were worked out for its potential use as a bioremediator of wastewater. The reduction in the amount of Cr⁶⁺ after 2, 4, 6 and 8 days of algal culture containing 5.0 μg Cr⁶⁺ ml⁻¹ of culture medium was 77, 85, 92 and 97%, respectively. Distigma could also remove 48% Cd²⁺after 2 days, 68% after 4 days, 80% after 6 days and 90% after 8 days from the medium. The heavy metal uptake ability of Distigma can be exploited for metal detoxification and environmental clean-up operations.     

Interaction of ceruloplasmin and 5-lipoxygenase

The interaction between ceruloplasmin (CP), the multicopper oxidase of human plasma, and 5-lipoxygenase (5-LO), the key enzyme of leukotriene synthesis, is shown for the first time. By Western-blotting and mass spectrometry of tryptic fragments, it is shown that 5-LO from protein extract of human leukocytes binds with immobilized CP. Dose-dependent influence of intact CP on leukotrienes synthesis is found: CP reduced leukotrienes synthesis in leukocytes in a dose above 50 μg/ml (normal CP concentration in plasma is about 300–400 μg/ml). Proteolyzed CP and apo-form of CP is unable to inhibit activity of 5-LO. CP increased activity of 5-LO at low doses (5–10 μg/ml). On the whole, the influence of CP on phagocytosis index of leukocytes coordinates with influence on activity of 5-LO: the index increased in the range of 2–10 μg/ml CP and decreased at doses of CP above 40 μg/ml. The dual role of CP in regulation of cellular response of leukocytes is discussed.     

Heart valve allograft decontamination with antibiotics: impact of the temperature of incubation on efficacy

Heart valve allografts are typically processed at 4°C in North America, including the step of antibiotic decontamination. In our own experience with heart valve banking, we often observe persistent positive cultures following decontamination at wet ice temperature. We hypothesized that warmer temperatures of incubation might increase the efficacy of the decontamination procedure. In a first series of experiments, 12 different bacterial species were grown overnight, frozen in standardized aliquots and used directly to inoculate antibiotic cocktail aliquots at 10⁵ colony-forming units (CFU)/ml. The antibiotic cocktail contains vancomycin (50 μg/ml), gentamicin (80 μg/ml) and cefoxitin (240 μg/ml) in Dulbecco’s Modified Eagle’s Medium. Inoculated aliquots were incubated at 4, 22 and 37°C and CFUs were determined at regular intervals up to 24 h post-inoculation. In a second set of experiments, 10 heart valves were spiked with 5000 CFU/ml and incubated with antibiotics at 4 and 37°C for 24 h. The final rinse solutions of these heart valves were filtered and tested for bacterial growth. After 24 h of incubation, CFUs of all 12 bacterial species were reduced by a factor of only one to two logs at 4°C whereas log reductions of 3.7 and 5.0 or higher were obtained at 22 and 37°C, respectively. Most microorganisms, including Staphylococcus epidermidis, Lactococcus lactis lactis and Propionibacterium acnes survived well the 24-h antibiotic treatment at 4°C (<1 Log reduction). All 10 heart valves that were spiked with microorganisms had positive final rinse solutions after antibiotic soaking at 4°C, whereas 8 out of 10 cultures were negative when antibiotic decontamination was done at 37°C. These experiments show that a wet ice temperature greatly reduces the efficacy of the allograft decontamination process as microorganisms survived well to a 24-h 4°C antibiotic treatment. This could explain the high rate of positive post-processing cultures obtained with our routine tissue decontamination procedure. Increasing the decontamination temperature from 4 to 37°C may significantly reduce the incidence of post-disinfection bacterial contamination of heart valves.     

Influence of lipopolysaccharides and lipids A from some marine bacteria on spontaneous and Escherichia coli LPS-induced TNF-α release from peripheral human blood cells

Some endotoxic properties of lipopolysaccharides (LPS) and lipids A (LA) from the marine bacteria Marinomonas communis ATCC 27118^T, Marinomonas mediterranea ATCC 700492^T, and Chryseobacterium indoltheticum CIP 103168^T were studied. The preparations tested were shown to have high 50% lethal doses (4 μg per mouse for LPS from M. mediterranea and more than 12 μg per mouse for two other LPS and LA from C. indoltheticum) and were moderate (371 ± 37 pg/ml at 10 μg/ml of C. indoltheticum LPS), weak (148 ± 5 pg/ml at 1 μg/ml of M. mediterranea LPS), and zero (LA and LPS from M. communis and LA from C. indoltheticum) inducers of tumor necrosis factor α (TNF-α) release from peripheral human blood cells. The capacity of the LA and LPS samples from marine bacteria to inhibit TNF-α release induced by LPS from Escherichia coli O55: B5 (10 ng/ml) was also studied. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 7, pp. 936–944.