Notes: For at least 30 years it has been well known that very small electric currents applied to various kinds of trauma have a beneficial therapeutic result for wounds. The most effective methods so far utilized some form of direct current, by inserting an electric lead on each side of the wound and passing a weak electric current across the wound. The drawback of this method is invasive. In this study, we evaluated the effects of a noninvasive electric magnetic field device, Field Therapy Accelerator or FTA (Advatech Corp.), on cell migration of human skin keratinocytes in an in vitro incitional wound model. Normal human epidermal keratinocytes were maintained in growth media and grow into confluent. At time 0, a cross-shaped wound gap or cell-free zone was made among confluent monolayer cells in the center of the culture dish. Cells were treated with FTA for 1 hour immediately after wounding and 1 hour every 24 hours for a total of 4 hours in 4 days. The cell cultures were examined at time 0 and every 24 hours thereafter. A Zeiss Axiovert 200 inverted microscope with a digital camera system was used to capture the images and an AxioVision 3.0 image analysis software was used to measure the gap area. Cell migration was quantified by the time and percentage of the wound gap covered by the cells that migrated in and filled the gap. The results showed that the FTA treatment significantly accelerates skin keratinocyte migration. The wound gaps completely filled 6 days after wounding, 3 days earlier than control (no treatment) group (P 〈 0.001). Cell migration is a key step leading to the reepithelialization or the restoration of skin structure. This study showed the strong effects of FTA on keratinocyte migration, suggesting one of mechanisms that electric magnetic field works on wound healing.

Notes: Purified homogenous glutamic acid decarboxylase (GAD) from mouse brain and rabbit antiserum prepared to partially purified GAD gave only one sharp precipitin band in the Ouchterlony double diffusion test. GAD activity was inhibited partially by incubating with the antiserum. The maximal extent of inhibition was approximately 50 per cent. In the presence of antiserum all enzyme activity could be precipitated. The precipitates formed by GAD and antiserum had about 50 per cent of the enzyme activity and the Km values for both glutamic acid and pyridoxal phosphate were significantly higher than those of the control system. Pyridoxal phosphate protected GAD from inhibition only slightly, even at very high concentrations. The results suggest that the antibodies may not react with the catalytic site, but rather that the inhibition of enzyme activity is attributable to indirect effects.

Notes: Abstract— The effects of divalent metal ions, sulfhydryl reagents, carbonyl trapping reagents, substrate analogs, and organic solvents on purified mouse brain 4-aminobutyrate-2-ketoglutarate transaminase (EC 2.6.1.19) and the subunit structure of this enzyme were studied. Of the metal ions tested, Hg2+ was found to be the most potent inhibitor inhibiting the enzyme 50 percent at a concentration of 0-7 μM. The order of decreasing inhibitory potency for the divalent metal ions was: Hg2+± Cd2+± Zn2+± Cu2+± Co2+± Ba2+± Sr2+± Ni2+± Mn2+± Ca2+± Mg2+. p-Chloromercuribenzoale was the most potent inhibitor among the sulfhydryl reagents tested inhibiting the enzyme to the extent of 50 per cent at 0-5 μM 3-Mercaptopropionic acid was found to be a competitive inhibitor for GABA and non-competitive for 2-ketoglutarate. The Ki, value was estimated to be 13 μM. Aminooxyacetic acid was the most potent inhibitor of the carbonyl trapping agents with a K, value of 0-06 μM. being competitive with GABA and non-competitive with 2-ketoglutarate. Hydroxylamine and hydrazine were the next most potent compounds in this group. Of a series of substrate analogs and metabolites tested, only acetic acid, propionic acid, butyric acid, glutamic acid, adipic acid, pimelic acid and 2-ketoadipic acid inhibited the enzyme to a significant extent. Dioxan inhibited the enzyme 50 per cent at a concentration of 5 per cent (v/v) whereas methanol and ethanol only inhibited 5-10 per cent at 10 per cent (v/v) concentration.A spectrum of the native enzyme at pH 7-2 showed maxima at 278 nm. 330 nm and 411 nm. Treatment of the enzyme with aminooxyacetic acid or 3-mercaptopropionic acid caused the maximum at 411 nm to disappear.Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed two protein bands. The molecular weights of these two subunits were determined to be 53.000 and 58,000, respectively.

Notes: —l-Glutamate decarboxylase purified from mouse brain was found to be highly sensitive to the sulfhydryl reagents, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) and p-chloromerburibenzoate (PCMB), which were competitive inhibitors (Ki for DTNB is 1·1 · 10−8m). Iodoacetamide and iodoacetic acid were less effective inhibitors than DTNB and PCMB. The mercapto acids, 3-mercaptopropionic, 2-mercaptopropionic, and 2-mercaptoacetic acids were potent competitive inhibitors with Ki values of 1·8, 53 and 300 μm, respectively. 2-Mercaptoethanol was less effective. Aminooxyacetic acid was the most potent carbonyl-trapping reagent tested inhibiting the enzyme activity completely at 1·6 μm, followed by hydroxylamine, hydrazine, semicarbazide, and d-penicillamine. Carboxylic acids with a net negative charge were strong competitive inhibitors e.g. d-glutamate (Ki 0·9 mm), α-ketoglutarate (Ki, l·2mm), fumarate (Ki,1·8 mm), dl-β-hydroxyglutamate (Ki, 2·8 mm), l-aspartate (ki, 3·1 mm) and glutarate (Ki, 3·5 mm). 2-Aminophosphonobutyric and 2-aminophosphonopropionic acids, phosphonic analogs of glutamate and aspartate, respectively, had no effect at l0mm. γ-Aminobutyric acid, l-glutamine, l-γ-methylene-glutamine, and α,γ-diaminoglutaric acid, amino acids with no net negative charge at neutral pH, had no effect at 5 mm. Glutaric and α-ketoglutaric acids were the most potent inhibitors among the various dicarboxylic and α-keto-dicarboxylic acids tested (Ki, 3·5 and 1·2 mm, respectively). Compounds with one carbon less, succinic and oxalacetic acids, or with one carbon more, adipic and α-ketoadipic acids, were less inhibitory. The monovalent cations, Li+, Na+, NH4+, and Cs+ had no effect on l-glutamate decarboxylase activity in concentrations up to 10mm. Divalent cations, on the other hand, were very potent inhibitors. Among eleven divalent cations tested, Zn2+ was the most potent inhibitor, inhibiting to the extent of 50 per cent at 10μm. The decreasing order of inhibitory potency was: Zn2+ 〉 Cd2+, Hg2+, Cu2+ 〉 Ni2+ 〉 Mn2+ Co2+ 〉 Ba2+ 〉 Ca2+ 〉 Mg2+ 〉 Sr+2, The anions, I−, Br−, Cl− and F− were only weak inhibitors. The Ki value for Cl− was 17mm. The above findings suggest minimally the presence of aldehyde, sulfhydryl and positively charged groups at or near the active site of the holoenzyme. Intermediates of glycolysis had little effect on l-glutamate decarboxylase activity, but intermediates of the tricarboxylic acid cycle, e.g. α-ketoglutarate (Ki= 1·2 mm) and fumarate (Ki= 1·8 mm) were relatively potent inhibitors. The nucleotides, ATP, ADP, AMP, cyclic AMP, GTP, GDP, GMP, and cyclic GMP were weak inhibitors. l-Norepinephrine (Ki= 1·3 mm) and serotonin were potent inhibitors, while acetylcholine, dopamine and histamine were less effective. Ethanol and dioxane inhibited the enzyme activity to the extent of 20-50 per cent at 10 per cent (v/v), while slight activation was observed at low concentrations (0·1-1 per cent) of both solvents. The possible role of Zn2+ and some metabolites in the regulation of steady-state levels of γ-aminobutyric acid also was discussed.

Notes: Electrophoretically and ultracentrifugally homogeneous glutamic acid decarboxylase purified from mouse brain showed multiple protein bands after electrophoresis in SDS polyacrylamide gel. The positions and intensities of the multiple bands were constant despite different treatments of the enzyme with various concentrations of SDS, β-mercaptoethanol, and urea at different temperatures. The major band had an apparent molecular weight of approximately 60,000 daltons and there were three minor bands of molecular weights, about 120,000, 90,000, and 75,000 daltons, respectively. The molecular weights of almost all bands were approximately integral multiples of 15,000. The possible subunit structure of this enzyme has been discussed in the light of the latter data and data previously reported from ultracentrifugation and gel filtration studies. We suggest that this enzyme may be a hexamer consisting of 15,000-dalton sub-units and that dissociation of these sub-units in SDS is accompanied by reassociation into a variety of aggregates, the probability of whose formation is determined by structural features that are more important than the differences encountered under the environmental conditions employed in these studies.

Notes: A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis-active Cry1Ac and API-B chimeric gene. Infected embryogenic calli were co-cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7-8 weeks. Then, the kanamycin-resistant calli (Km1) subcultured in proliferation medium would re-differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100-ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty-five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API-B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium-mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.

Notes: In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 °C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 °C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 °C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 °C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 °C) compared with the non-permissive temperature of 28 °C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis.