In vitro shoot regeneration from leaf mesophyll protoplasts of hybrid poplar (Populus nigra x P. maximowiczii)

Summary Protoplasts were isolated from leaf mesophyll of hybrid poplar (Populus nigra X P. maximowiczii) with a mean yield of 10.4 x 10⁶ protoplasts per g fresh weight using 2.0% Cellulase Onozuka R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, and 0.05% Pectolyase Y-23 with CPW salts solution containing 0.6 M mannitol, 0.002 M DTT, 3 mM MES at pH 5.6. A liquid plating method produced the highest frequency of dividing protoplasts (48.6%) using an MS medium without NH₄NO₃. The highest percent of colony formation was 22.8%, produced with fabric supported semi-solid (0.5% w/v) agar plating method using the same culture medium. Growing cell colonies and/or micro-calli were transferred to a fresh semisolid agar medium containing 0.44 M BAP and 9.0 M 2,4-D. Multiple shoots were produced from protoplast-derived callus after culture on MS medium containing 6.8 M zeatin. After root induction on half-strength MS medium that lacked growth regulators, shoots were transferred to pots containing artificial soil mix.Abbreviations CPW Cell and Potoplast Wash solution - LPM Liquid Plating Method - LDM Liquid Drop Method - HDM Hanging Drop Method - FSPM Fabric supported Semi-solid agar Plating Method - DTT Dithiothreitol - MES 2-(N-morpholino) ethane sulfonic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - MS Murashige and Skoog (1962)     

Characterization of D2 receptors and dopamine levels in the thalamus of the rat.

We kinetically characterized D2 receptors in thalami pooled from a group of Sprague-Dawley rats and then determined thalamic levels of dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and norepinephrine (NE) in relation to a measure of thalamic DA D2 receptor densities in another group of rats. The equilibrium dissociation constant (kd) was estimated as 0.1 nM by three independent methods, while the Bmax for thalamic D2 receptors was found to be 6.4 fmol/mg p using 3H-spiperone as ligand and ketanserin to occlude 5HT2 binding. Kinetic constants were in agreement with previously reported kinetic data from rodent caudate-putamen. This suggests that thalamic D2 receptors are similar to D2 receptors from other brain areas. Mean thalamic levels of DA (22.6 ng/mg p), DOPAC (1.19 ng/mg p) and HVA (0.31 ng/mg p) concur with previous reports of a sparse distribution of thalamic DA neurons. D2 receptor densities were positively correlated with DA metabolites DOPAC (P less than .05; r = 0.423) and HVA (P less than .05; r = 0.368), but not DA or NE. These results establish fundamental characteristics of thalamic DA neurotransmission to assist in the investigation of behavioral pharmacology of this area.     

Lysophosphatidylserine enhances the transfer of 22:6n3 to lysophosphatidic acid in rat brain microsomes.

Although the acyl groups of phosphatidylserine in brain are uniquely enriched in docosahexaenoic acid (22:6n3), the mechanism for this enrichment is not well understood. When rat brain homogenates and microsomes were incubated in the presence of lysophosphatidylserine (LPS) together with [14C]22:6n3 and cofactors for activation to its acylCoA, very little radioactivity was incorporated into phosphatidylserine (PS). On the other hand, [14C]20:4n6 was more actively incorporated into PS. Addition of LPS (1-10 uM), however, resulted in a 2-5 fold enhancement of the transfer of labeled 22:6n3 and 20:4n6 to phosphatidic acid (PA). Kinetic analysis indicated the ability of LPS to lower the Km and increase the Vmax of the lysophosphatidic acid (LPA) acyltransferase reaction. Among other lysophospholipids tested, lysophosphatidylserine was most effective in enhancing PA biosynthesis. Since PA is an important intermediate for de novo biosynthesis of phospholipids, these results reveal a novel mechanism for promoting synthesis of PA enriched in polyunsaturated fatty acids in brain.     

Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody.

A human monoclonal antibody designated 15e is reactive with the envelope glycoprotein (gp120) of multiple isolates of human immunodeficiency virus type 1 (HIV-1). Antibody 15e also neutralizes HIV-1 with broad specificity and blocks gp120 binding to CD4. Characterization of the 15e epitope shows that it is conformation dependent and is distinct from previously recognized functional domains of gp120, suggesting that this epitope represents a novel site important for HIV-1 neutralization and CD4 binding. These findings have implications for the development of a vaccine for AIDS.     

Isolation and Characterization of a UDPGlucose: Flavonol O-Glucosyltransferase from Illuminated Red Cabbage (Brassica oleracea cv Red Danish) Seedlings

A UDPGlc:flavonol O³-glucosyltransferase (EC 2.4.1.91) that catalyzes the formation of quercetin and kaempferol O³-glucosides has been purified about 1450-fold from illuminated red cabbage (Brassica oleracea cv Red Danish) seedlings with a 3.3% yield. Purification of the enzyme was achieved by (NH₄)₂SO₄-precipitation, gel-filtration, ion-exchange chromatography on DEAE-Bio-Gel and Q-Sepharose, chromatofocusing, and electrophoresis in nondenaturing polyacrylamide (10%) gels. The enzyme preparation had a pH optimum between 5.8 and 6.2, isoelectric point in the pH range 4.25 to 4.55, a M_r of 59,000, and it was composed of two similar subunits of M_r 29,500. The glucosyltransferase reached half substrate saturation at 180 micromolar (UDPGlc) and 7 micromolar (quercetin) concentrations. Kaempferol, which was glucosylated at a relative rate of 87%, had a lesser affinity for the enzyme (K_m~12 micromolar). Flavanones, flavanols, flavones, dihydroflavonols, and anthocyanidins were not readily utilized as substrates, suggesting that the enzyme is specific for flavonol glucoside biosynthesis.     

Biocompatibility of radiolucent breast implants

Current implants for breast augmentation containing silicone gel, saline, or both are radiopaque on mammographic examination and can totally obscure microcalcifications and soft-tissue masses. The effect of these implants on the detection of early breast cancers in patients who have undergone augmentation mammaplasty remains unproven and controversial. Implants filled with medium-chain triglycerides (peanut oil) are radiolucent on mammographic examination and allow visualization of both soft-tissue masses and microcalcifications. To investigate the biocompatibility of radiolucent implants, 10 cc of sterile, nonpyrogenic peanut oil was injected subcutaneously into rats using silicone gel as a control. Twenty-one rabbits had two 125-cc silicone shell implants inserted on either side of the chest wall. The right-sided shell was filled with 125 cc of sterile saline, and the left-sided shell was filled with 125 cc of sterile, nonpyrogenic peanut oil. Results were determined by both histologic and radiographic examination. Rats injected with peanut oil equivalent to 7 percent of their body weight rapidly absorbed the freely injected oil without detriment. Histologic examination of the lungs, liver, kidneys, and tissues adjacent to the injection sites demonstrated no abnormalities. There was no evidence of allergic, toxic, inflammatory, or neoplastic response. Eighteen of 21 rabbits survived more than 3 months. Radiographs showed the oil-filled implants to be radiolucent, whereas the saline-filled controls obscured the surrounding soft and bony tissues. Histologic examination demonstrated a fibrous capsule surrounding both types of implants. Histologic examination of the lungs, liver, and kidneys showed no significant abnormalities. These and previous studies have shown peanut oil to be biocompatible when freely injected either intramuscularly or subcutaneously. This study demonstrates that a radiolucent, peanut oil-filled implant is biocompatible in animals and that further long-term studies for its use in humans are merited.     

Oxygen transport and peripheral microcirculation in long-term diabetes

The purpose of this investigation was to evaluate the impact of long-term diabetes on muscle blood flow (MBF) and oxygen transport (vO2) during exercise. Twelve male patients (58 +/- 8 years, mean +/- SD), with at least a 10-year history of diabetes controlled by insulin, and seven age-matched controls (56 +/- 5 years, mean +/- SD) participated in this study. No patient had been clinically diagnosed as having peripheral vascular disease, and on the average resting ankle/arm systolic blood pressure ratios were normal. Following a baseline period, 5 min of cycle ergometer exercises at 75 W were performed in the upright position and, after 1-hr recovery, in the supine position. Continuous vO2 was determined via breath-by-breath analysis. MBF was measured in the vastus lateralis (VL) and tibialis anterior (TA) by 133Xe clearance. In the erect position, the diabetic group (compared with the control group, respectively) exhibited significantly (P less than 0.05) lower exercise MBF [ml. (100 g.min)-1] in both VL (19 +/- 2.5 vs 30.9 +/- 2) and TA (13.7 +/- 2 vs 22.0 +/- 4), a lower steady-state VO2 (1.3 +/- 0.3 vs 1.7 +/- 0.2 liters.min-1) during exercise including the values in the last 15 sec of exercise, and greater accumulation of blood lactate (35 +/- 2 vs 22.0 +/- 2 mg/100 ml). The same trends in the data were observed during supine exercise; however, the blood pressure of the diabetics was significantly elevated during exercise when compared with that of controls. The reduced exercise MBF in the TA and VL demonstrated that impaired microvascular flow, without clinically overt peripheral vascular disease, in long-term diabetics leads to reduced oxygen delivery and exercise tolerance.     

Field uniformity of the Japonica rice region of Taiwan as estimated by relative genetic contribution

Summary Despite the concerns for genetic vulnerability that were raised in the 1970s, the field uniformity of the Japonica rice (Oryza sativa L.) region in Taiwan has increased since 1980 with over 82% of the cultivated areas being covered by as few as three varieties and over half of this hectarage by a single variety. Japanese plant introductions are the major ancestral contributors of genetic constituents for varieties released in Taiwan. The main constitution of the genetic base present in the field has changed little since 1971. Six common ancestors comprised 60%, 55%, 78%, and 77% of the genetic constituents present in the field in 1971, 1976, 1981, and 1986, respectively. These estimates revealed that at least 55% of the genes utilized in the last 15 years came from the same sources. Recent efforts in introducing new germ plasm sources to variety development should continue to alleviate the possible crop loss due to continuous monoculture.Research supported by National Science Council (NSC 78-0211-B005-14)     

Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures

Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene was correlated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures. However, m-xylene was degraded by a subculture that was incubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.