REGENERATION AND SEXUAL DIFFERENTIATION OF GRIFFITHSIA JAPONICA (CERAMIACEAE,RHODOPHYTA) THROUGH SOMATIC CELL FUSION1

Hybrid cells were obtained from somatic cell fusion among male, female, and tetrasporangial plants in Griffithsia japonica Okamura by a wound-healing process. Isolated fusion cells regenerated new mature plants with mixed reproductive structures. The plants regenerated from hybrid cells between male and female plants developed into 1) spermatangiate, 2) carpogonial, 3) bisexual with spermatangia and carpogonial branches, 4) mixed-phase with spermatangia and tetrasporangia, or 5) bisexual/mixed-phase plants with spermatangia, carpogonial branches, and tetrasporangia. About 70% of the plants regenerated from hybrid cells between male and female plants produced tetrasporangia that were always formed with spermatangia on a single cell. Some of those tetrasporangia released tetraspores, six of which gave rise to mature plants. The plants regenerated from hybrid cells between male and tetrasporangial plants developed into spermatangiate, tetrasporangiate, or mixed-phase plants with spermatangia and tetrasporangia. The plants regenerated from hybrid cells between female and tetrasporangial plants developed into carpogonial, tetrasporangiate, or mixed-phase plants with carpogonial branches and tetrasporangia. All types of reproductive structures we re functional.     

ACCUMULATION OF ASTAXANTHIN IN HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)1

In N-limited continuous chemostat cultures of the green alga Haematococcus lacustris (Gir.) Rostaf. (UTEX 16), the steady-state astaxanthin content of the cells was determined by the specific growth rate of the cultures. The highest, pigment content was obtained at the lowest dilution rate. The specific rate of astaxanthin accumulation was, however, a function of the photon flux density measured at the illuminated culture surface. In nongrowing Haematococcus cultures, the specific rate of astaxanthin accumulation was determined by the growth rate of the culture during growth phase. The highest possible cellular astaxanthin content of all cultures was comparable and independent of the culture parameters.     

Supercooling and cold-hardiness in eggs of western and northern corn rootworms

Oviposition by northern corn rootworms, Diabrotica barberi Smith and Lawrence, and western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), key pests of corn in the Great Plains of the USA, occurs in the soil during late summer. Overwintering eggs are exposed to variable soil moisture and temperatures below ?5 °C. The winter mortality of eggs in the soil is a primary factor that determines the potential for larval injury to corn the following spring. Our studies aimed to determine the comparative supercooling capacities of northern and western corn rootworm eggs and to assess egg mortality following brief exposure to extreme low temperature, ranging from ?12.0 to ?21.5 °C, under three moisture regimes. Eggs of northern corn rootworm were supercooled to a temperature as low as ?27 °C, and survived supercooling to a greater extent than did western corn rootworm eggs. Moisture treatment prior to supercooling had little effect on northern corn rootworm eggs. Western corn rootworm eggs were more resistant than northern corn rootworm eggs to the effects of desiccation followed by supercooling. The survival of northern corn rootworm eggs was better than western corn rootworms under dry conditions, followed by exposure to temperatures of ?12.0 and ?17.5 °C, but was very low at ?21.5 °C, regardless of the moisture regime. The results suggest that moisture and temperature may interact in the soil environment to determine the overwintering survival of corn rootworms. It is evident from these studies that both rootworm species experience mortality at temperatures well above the supercooling points of the eggs, but that differences exist in the effects of substrate moisture treatments on the cold‐hardiness of eggs from the two species.     

Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Cigarette smoke-induced bronchoconstriction: causative agents and role of thromboxane receptors

Hong, Ju-Lun, and Lu-Yuan Lee. Cigarette smoke-inducedbronchoconstriction: causative agents and role of thromboxane receptors. J. Appl. Physiol. 81(5):2053-2059, 1996.Inhalation of cigarette smoke induces a biphasicbronchoconstriction in guinea pigs: the first phase is induced by acombination of cholinergic reflex and tachykinins, whereas the secondphase involves cyclooxygenase metabolites (J.-L. Hong, I. W. Rodger,and L.-Y. Lee. J. Appl. Physiol. 78:2260-2266, 1995). This study was carried out to further determinethe causative agents in the smoke and the types of prostanoid receptorsand endogenous prostanoids mediating the bronchoconstriction. Inhalation of 10 ml of high-nicotine cigarette smoke consistently elicited the biphasic bronchoconstriction in anesthetized and artificially ventilated guinea pigs. Pretreatment with hexamethonium (10 mg/kg iv) significantly reduced the first-phase bronchoconstriction but did not have any measurable effect on the second-phase response. Insharp contrast, gas-phase smoke did not elicit any bronchoconstrictive effect. Furthermore, when the animals were challenged with low-nicotine cigarette smoke, only a single second-phase response was evoked, accompanied by increases in thromboxane (Tx)B₂ (a stable metabolite ofTxA₂), prostaglandin (PG)D₂,PGF₂ in the bronchoalveolar lavage fluid. The bronchoconstrictive response induced by low-nicotine smoke was completely prevented by pretreatment with SQ-29548 (0.3 mg/kgiv), a TxA₂-receptor antagonist.These results indicate that 1)nicotine is the primary causative agent responsible for the first-phasebronchoconstriction and 2)nonnicotine smoke particulates evoke the release ofTxA₂,PGD₂, andPGF₂, which act onTxA₂ receptors on airway smoothmuscles and induce the second-phase response to cigarette smoke.

     

Penicillin acylase-catalyzed synthesis of β-lactam antibiotics in water-methanol mixtures: effect of cosolvent content and chemical nature of substrate on reaction rates and yields

The synthesis of four β-lactam antibiotics (penicillin G, pivaloyloxymethyl ester of penicillin G, ampicillin and pivampicillin) catalyzed byEscherichia coli penicillin acylase has been investigated in water-methanol mixtures. The enzyme reactions were either thermodynamically or kinetically controlled at the same conditions using phenylacetic acid andd--phenylglycine methyl ester as acyl donors and 6-aminopenicillanic acid and pivaloyloxymethyl 6-aminopenicillanic acid as acyl acceptors. It has been found that the influences of the cosolvent content on the reaction rates and synthetic yields are significantly different depending on the substrates used in the experiments. On the other hand, within certain ranges of the methanol content (up to ca. 40% (v/v) the residual activities of the enzymes in water-methanol mixtures were only slightly lower than those in aqueous media. To analyze the factors that determine the reaction rate in water-cosolvent mixtures, the effect of methanol on the apparent pK values of the substrates has been investigated, and a mathematical model has been developed on the basis of the assumption that the enzyme binds non-ionized substrates. Model simulation results indicate that the solvent effect on reaction rates is mainly attributed to the kinetic effects of changes in apparent pK values.     

Determination of nalbuphine by high-performance liquid chromatography with ultraviolet detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with ultraviolet detection was developed for the determination of nalbuphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane-isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of nalbuphine and ethylmorphine (internal standard) were greater than 86%. Calibration graphs were linear over the concentration range 0.75–150 ng/ml with a coefficient of variation, both within-day and between-day, of less than 10% at any level. The limit of quantitation was 0.75 ng/ml of plasma based on a signal-to-noise ratio of 3. Seven other clinically used analgesics were investigated to check for potential interferences and their analytical conditions. The specificity of this assay was checked with a metabolite of nalbuphine (noroxymorphine). Nalbuphine in plasma did not decompose significantly at −20°C for six weeks. Pharmacokinetic application in three surgical patients and four rabbits revealed that nalbuphine followed a linear three-compartment model with two distribution phases. The two distribution and one elimination half-lives and the plasma clearance of nalbuphine were 0.9, 5.8 and 157 min and 370 ml/min in human, and 3.5, 28 and 117 min and 21 166 ml/min in rabbits.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

A simple model of human foveal ganglion cell responses to hyperacuity stimuli

We developed a physiologically plausible model of the first steps of spatial visual information processing in the fovea of the human retina. With the predictions of this model we could support the hypothesis that, for moderate contrasts ( 40%), hyperacuity is mediated by the magnocellular (MC-) pathway. Despite the lower sampling density in the MC pathway, as compared to the parvocellular (PC-) pathway, the information that is transferred by the MC ganglion cells is sufficient to achieve thresholds comparable to those of human subjects in psychophysical tasks. This is a result of the much higher signal-to-noise ratio of the MC pathway cell signals. The PC pathway cells do not transfer enough information for hyperacuity thresholds.