A single-stage two-flap method of total ear reconstruction

A single-stage two-flap method of total ear reconstruction in congenital microtia is reported. This method was derived from the one-stage reconstruction described by Song and Song. Two flaps defined by vascular basis were elevated on the mastoid area: the superficial skin flap supplied mainly by subcutaneous pedicled arteriole perforators from the posterior auricular artery and the deeper axial-pattern fascial flap including the posterior auricular artery itself. The ear framework, exaggeratedly carved using autologous rib cartilage, could be inserted easily between the two flaps, simultaneously producing the auriculocephalic angle and the conchal wall. Intraoperative expansion of the skin flap and postoperative external ear molding also were performed to create aesthetically pleasing ears.     

Elongation and termination reactions of protein synthesis on maize root tip polyribosomes studied in a homologous cell-free system

We show that the control of gene expression at the level of elongation and termination of protein synthesis can be observed in vitro. Free cytoplasmic polyribosomes were isolated from maize (Zea mays) root tips, and translated in root tip extracts that had been fractionated with ammonium sulfate to contain elongation factors, and be depleted in initiation factors. The root tip extract performs elongation and termination reactions as efficiently as wheat germ extracts. The translation products of the maize system are the same as made in vivo. The dependence of these in vitro elongation and termination reactions on pH was determined. Total protein synthesis in this system exhibits an optimum at pH ~7.5. However, the pH dependence of rates of synthesis of individual proteins is not at all uniform; many polyribosomes become stalled when translated at low pH. These data were compared with the elongation and termination capacity of polyribosomes isolated from oxygenated and hypoxic root tips (tissue having, respectively, high and low cytoplasmic pH values). We observed an inverse relationship between the relative abundance of many specific translatable mRNAs in polyribosomes of hypoxic root tips, and the relative rates of elongation and termination reactions on the different mRNAs at low pH in vitro. These results suggest that changes in intracellular pH in hypoxic root tips can be sensed directly by the translational machinery and thereby selectively modulate gene expression.     

Differences in thermotolerance induced by heat or sodium arsenite: correlation between redistribution of a 26-kDa protein and development of protein synthesis-independent thermotolerance in CHO cells

In previous studies, we have demonstrated the differences in thermotolerance induced by heat and sodium arsenite (Lee et al., Radiat. Res. 121, 295-303, 1990). In this study, we investigated whether a 26-kDa protein might play an important role in evincing these differences. Chinese hamster ovary (CHO) cells treated for either 1 h with 100 microM sodium arsenite (ARS) or 10 min at 45.5 degrees C became thermotolerant to a test heat treatment at 43 degrees C administered 6 or 12 h later, respectively. After the test heating at 43 degrees C for 1.5 h, the level of 26-kDa protein in the nucleus was decreased by 92% in nonthermotolerant cells, 78% in ARS-induced thermotolerant cells, and 3% in heat-induced thermotolerant cells. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) after ARS treatment eliminated thermotolerance to 43 degrees C and delayed restoration of the 26-kDa protein in the nucleus. In contrast, CHM neither prevented the development of thermotolerance nor inhibited the restoration of the 26-kDa protein in heat-induced thermotolerant cells. However, when cells were exposed to cold (4 degrees C), immediately after initial heating, restoration of the 26-kDa protein and development of thermotolerance did not occur. These results demonstrate a good correlation between the restoration and/or the presence of this 26-kDa protein and the development of protein synthesis-independent thermotolerance.     

Anaerobic degradation of toluene by a denitrifying bacterium

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Characteristics of a novel high viscosity polysaccharide,methylan, produced byMethylobacterium organophilum

Summary A new anionic high molecular weight polysaccharide, Methylan, was produced byMethylobacterium organophilum from methanol as a sole source of carbon and energy under the specific culture conditions. By GPC and light scattering, the molecular weight was determined to be 2–4×10⁶ dalton and the distribution of molecular weight was very homogeneous. Methylan was composed of carbohydrate (80%), uronic acid (12%), protein (6%) and pyruvic acid (5%). The sugar composition of Methylan was identified as glucose, galactose and mannose with the approximate molar ratio of 232. Methylan solution showed a pseudoplastic non-Newtonian fluid property at the concentration above 0.05%. At the concentration of 1% Methylan solution, the consistency index was 18,000 centipoise which was almost 10 times higher than that of Xanthan and the flow behavior index was 0.15.     

High level expression of human tissue-type plasminogen activator gene in mouse C127 cell.

We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.     

Artificial Induction and Evaluation of a Mild Isolate of Tomato Spotted Wilt Virus

A severe isolate (BL) of tomato spotted wilt virus (TSWV) that originated from Hawaii was treated with nitrous acid in an effort to obtain mild mutants. The standardized procedure used in mutation experiments was: extracting infected Gomphrena globosa L. leaf tissue in 0.01 M Na₂SO₃, 0.125 M sodium acetate and 0.4 M sodium nitrite at pH 5.5 and incubating the extract for 20 min at room temperature. The extract was inoculated to tobacco (Nicotiana tabacum L. cv. Havana 423) and local lesions were subsequently transferred to lettuce (Lactuca sativa L. cv. Minetto). One isolate (R27G) that incited mild symptoms in lettuce was obtained out of 868 local-lesion-transfers. Under greenhouse conditions, the isolate induced mild symptoms on tomato (Lycopersicon esculentum Mill.) but was severe on peppers (Capsicum annuum L.). The effect of the R27G isolate on growth of potted tomatoes kept outdoors was variable. In one trial, only 15 % of the fruit had symptoms versus 67 % in another trial. R27G fully protected Datura stramonium L. plants that were challenge inoculated with the severe parent BL isolate. Less effective cross protection was observed against a severe isolate from Oklahoma.     

Identification and characterization of an {alpha}-mannosidase from Trypanosoma cruzi

In this report we describe the first purification and characterizationof the acid -mannosidase from the human parasite Trypanosomacruzi. The purified enzyme exhibited a native mol. wt of 240000 Da and is apparently composed of four identical subunitsof mol. wt 58 000 Da. Each of the four subunits contains oneN-linked high-mannose-type oligosaccharide. The -mannosidaseexhibited a pH optimum of 3.5 and a pI of 5.9. This low pH optimumand the ability of swainsonine to inhibit its activity suggestthat the -mannosidase is a lysosomal enzyme. Antibodies againstthe T.cruzi enzyme did not react with mammalian lysosomal -mannosidaseand, conversely, antibody against a rat lysosomal -mannosidasedid not react with the T.cruzi enzyme. Thus, the T.cruzi enzymeappears to be distinct from its mammalian counterpart. -mannosidase lysosomal enzyme Trypanosoma cruzi