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.     

Correlation between fibronectin and its receptor in chick myoblast differentiation

Alterations in the amount of fibronectin and in the number of its receptors during myoblast differentiation of chicken embryo were investigated. The amount of fibronectin in the cell surface pool as measured by immunoblotting decreased during myogenesis To identify and characterize the fibronectin receptors on the myoblasts, the interactions of the 28,000 dalton (28 kDa) amino terminal fragment and 85,000 dalton (85 kDa) cell-binding fragment of fibronectin with my-oblasts were examined. The binding of the 28 kDa fragment was found to be time-dependent and reached a maximum level within 60 min. The unlabeled 28 kDa fragment inhibited the binding of the radioiodinated 28 kDa fragment, whereas the unlabeled 85 kDa fragment and antibody to integrin did not inhibit it, suggesting that the 28 kDa fragment interacts with the matrix assembly receptors but not with the cell adhesion receptors. There was a single class of 3.4 × 10⁵ binding sites per cell with an apparent dissociation constant of 1.4 × 10^?7 M on 30 hr old myoblasts. The specific binding of the radioiodinated 28 kDa fragment to myoblasts decreased as the fusion proceeded. This decrease of binding was consistent with the decrease in the amount of fibronectin. Furthermore, the levels of fibronectin and binding of the radioiodinated 28 kDa fragment in the fusion-blocked myoblasts by EGTA treatment appeared to remain constant. These results suggest that the decrease and/or loss of fibronectin during myoblast fusion is closely correlated with the alteration of fibronectin receptors and with the fusion of myoblasts.     

Nicardipine inhibits axon conduction but causes dual changes of acetylcholine release in the mouse motor nerve

The effects of nicardipine, a dihydropyridine Ca2(+)-channel antagonist, on neuromuscular transmission and impulse-evoked release of acetylcholine were compared with those of nifedipine. In the isolated mouse phrenic nerve diaphragm, nicardipine (50 microM), but not nifedipine (100 microM), induced neuromuscular block, fade of tetanic contraction, and dropout or all-or-none block of end-plate potentials. Nicardipine had no significant effect on the resting membrane potential and the amplitude of miniature end-plate potentials but increased the frequency and caused the appearance of large size miniature potentials. The quantal contents of evoked end-plate potentials were increased. In the presence of tubocurarine, however, nicardipine depressed the amplitude of end-plate potentials. The compound nerve action potential was also decreased. It is concluded that nicardipine blocks neuromuscular transmission by acting on Na+ channels and inhibits axonal conduction. Nicardipine appeared to affect the evoked release of acetylcholine by dual mechanisms, i.e., an enhancement presumably by an agonist action on Ca2+ channels, like Bay K 8644 and nifedipine, and inhibition by an effect on Na+ channels, like verapamil and diltiazem. In contrast with its inactivity on the amplitude of miniature end-plate potentials, depolarization of the end plate in response to succinylcholine was greatly depressed. The contractile response of baby chick biventer cervicis muscle to exogenous acetylcholine was noncompetitively antagonized by nicardipine (10 microM), but was unaffected by nifedipine (30 microM). These results may implicate that nicardipine blocks the postsynaptic acetylcholine receptor channel by enhancing receptor desensitization or by a use-dependent effect.     

ON THE ORIGIN OF INCIPIENT REPRODUCTIVE ISOLATION: THE CASE OF DROSOPHILA ALBOMICANS AND D. NASUTA

The nasuta subgroup of Drosophila consists of 12 known species classified within the immigrans group. D. nasuta and D. albomicans are two sibling species widely distributed throughout the Indo-Pacific tropics, which, although morphologically indistinguishable, have different meta-phase-chromosome configurations: chromosomes X and 3 are attached in D. albomicans, so that about 60% of its genes are sex-linked. Our experiments show that, at least in the laboratory, there is no sexual, mechanical, or gametic isolation between the two species. There is, however, hybrid “breakdown” expressed in three ways: 1) reduction in the number of F₂ hybrids produced per culture; 2) reduction in the fertility of F₂ (males) and F₃ (males and females) hybrid progenies; and 3) abnormal sex ratios in the progenies of crosses between strains of certain localities. In experimental populations, the karyotypes of both species are still present in substantial frequencies after 20 generations, although the frequencies of the two karyotypes vary depending on the geographic origin of the strains. Our results support the hypothesis that, in allopatry, the evolution of postzygotic isolation precedes that of prezygotic isolation. The mtDNA is polymorphic in both D. nasuta and D. albomicans and fairly similar between them. Assuming typical rates of mtDNA evolution, the two species would have diverged from each other about 500,000 years ago, whereas the African and Indian populations of D. nasuta (considered to be different subspecies by some authors) might have diverged some 350,000 years ago.     

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.     

Predation impact of Leptodora kindtii on population dynamics and morphology of Bosmina fatalis and B. longirostris in mesocosms

1. We assessed the impact of predation by the invertebrate predator Leptodora kindtii on Bosmina longirostris and B. fatalis, which show seasonal and reciprocal succession patterns in Lake Suwa, in a mesocosm experiment using 20‐L tanks with or without the predator under different food conditions. We also analysed morphological responses of the two Bosmina species to the predator in the tanks. 2. Bosmina fatalis dominated B. longirostris regardless of predator presence under high food density. However, the presence of Leptodora induced the dominance of B. fatalis more rapidly than its absence. On the contrary, no dominance of B. fatalis was observed in tanks with low food density, irrespective of the presence of the predator. Only B. fatalis showed morphological changes in response to the presence of Leptodora. 3. Mucrone length and antennule shape (angle between body and antennule and angle between antennules) showed marked responses at both high and low food densities, but antennule length responded only at high food density. Mucrone length seems to be a more effective defence against Leptodora. 4. The results suggest that B. fatalis is a superior competitor against B. longirostris and is more resistant to Leptodora predation, especially in good food conditions. The repeatedly observed seasonal succession of the two Bosmina species in the eutrophic Lake Suwa – the replacement of B. longirostris by B. fatalis following the occurrence of abundant Leptodora– seems to be caused by the selective predation of Leptodora on B. longirostris as well as the competitive ability of B. fatalis.     

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.     

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.