Polyhydroxyalkanoate (PHA) accumulation potential and PHA-accumulating microbial communities in various activated sludge processes of municipal wastewater treatment plants.

AIMS: To clarify the polyhydroxyalkanoate (PHA) accumulation potential and the PHA-accumulating microbial community structure in activated sludge in municipal wastewater treatment plants (WWTPs) and to identify their influential factors. METHODS AND RESULTS: Nine activated sludge samples were collected from municipal WWTPs employing various biological treatment processes. In acetate-fed 24-h batch experiments under aerobic and nitrogen- and phosphorus-limited conditions, polyhydroxybutyrate (PHB) content of activated sludge increased from 0-1·3 wt% to 7·9-24 wt%, with PHB yields of 0·22-0·50 C-mol 3-hydroxybutyrate (C-mol acetate)(-1). Microbial community analyses found that activated sludge samples that accumulated >20 wt% of PHB after 24-h PHA accumulation experiments had >5·0 × 10(8) copies g(-1)-mixed liquor-suspended solid of phaC genes. CONCLUSIONS: Results indicated that (i) activated sludge in municipal WWTPs can accumulate up to approx. 20 wt% of PHA without enrichment processes, (ii) PHA accumulation potential of activated sludge varied depending on the operational conditions (treatment processes) of WWTPs, and (iii) phaC gene number can provide a simple indication of PHA accumulation potential. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to compare the PHA accumulation potential and PHA-accumulating microbial communities in activated sludge of various treatment processes. Our findings may be useful for enhancing the resource recovery potential of wastewater treatment systems.

Apoptosis induced by arsenic trioxide in leukemia U937 cells is dependent on activation of p38, inactivation of ERK and the Ca2+-dependent production of superoxide.

The mechanism of the induction of apoptosis by arsenic trioxide (As2O3), which was demonstrated recently to be an effective inducer of apoptosis in patients with leukemia, was examined in detail in human leukemia U937 cells. Upon treatment of U937 cells with 50 microM of As2O3, complete inactivation of the kinases ERK1 and ERK2 was detected within 30 min. p38 was activated within 3 hr, and the maximum activity was detected at 6 hr, when DNA fragmentation remained undetectable. Experiments with transfected cells that expressed constitutively activated MEK1 and a specific inhibitor of p38 also suggested that inactivation of ERKs and activation of p38 might be associated with the induction of apoptosis by As2O3. In contrast to the inactivation of ERKs and the activation of p38, activation of JNK by As2O3 appeared to protect cells against the induction of apoptosis. Treatment of U937 cells with As2O3 also caused the Ca2+-dependent production of superoxide and intracellular acidification and a decrease in the mitochondrial membrane potential at the early stages of induction of apoptosis by As2O3. These changes preceded the release of cytochrome c from mitochondria and the activation of caspase-3. It should be possible to exploit the unusual characteristics of the mechanism of induction of apoptosis by As2O3 in U937 cells by making use of synergistic effects of this compound with other inducers of apoptosis.

Liquid chromatographic determination of oxytetracycline in swine tissues.

A simple and rapid method was developed for determination of oxytetracycline (OTC) in swine muscle and kidney by liquid chromatography (LC). The method involved homogenization of sample in acetonitrile-1M imidazole buffer containing 10 mM disodium ethylenediaminetetraacetic acid (Na2.EDTA) and 50 mM magnesium acetate (15 + 85) with added hexane, centrifugation, removal of the hexane phase, and ultrafiltration of the supernatant. L-column ODS (150 x 4.6 mm) with a mobile phase of acetonitrile-1M imidazole buffer containing 50 mM magnesium acetate and 10 mM Na2.EDTA (10 + 90) was used for the LC separation. A fluorescence detector was used at an excitation wavelength of 380 nm and an emission wavelength of 520 nm. The calibration graph was linear from 1.25 to 200 ng OTC. Recoveries of OTC from swine tissue fortified at levels of 0.05-1.0 microgram/g ranged from 58.0 to 67.3%. The quantitation and detection limits were 0.05 and 0.04 microgram/g, respectively.

Isolation of new temperature-sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation.

We have isolated two temperature-sensitive Saccharomyces cerevisiae mutants which exhibit a deficiency in mannose outer chain elongation of asparagine-linked oligosaccharide. The size of yeast glycoprotein, secretory form of invertase, of one mutant (och1) was slightly larger than that of the sec18 mutant at the non-permissive temperature, while that of the other mutant (och2) was almost the same as that of the sec18 mutant. Unlike sec mutants, the och mutants were not deficient in secretion of invertase. The och1 mutant showed a 2+:2- cosegregation with regard to the temperature sensitivity and mannose outer chain deficiency, suggesting that a single gene designated as OCH1 is responsible for these two phenotypes. The och1 mutant stopped its growth at the early stage of bud formation and rapidly lost its viability at the non-permissive temperature. The och1 mutation was mapped near the ole1 on the left arm of chromosome VII. The och1 mutant cells accumulated the external invertase containing a large amount of core-like oligosaccharides (Man9-10GlcNAc2) and a small amount of high mannose oligosaccharides (greater than Man50GlcNAc2) at the non-permissive temperature. Production of the active form of human tissue-type plasminogen activator was increased in the och1 mutant compared with the parental strain, suggesting the potential advantage of this mutant for the production of mammalian-type glycoproteins which lack mannose outer chains in yeast.

Neuronal organization of rat thalamus for processing information of vibrissal movements.

Vibrissa-responding neurons were searched for in the somatosensory part of the thalamic reticular nucleus (S-TR) and in the ventrobasal nucleus (VB) in urethane-anesthetized rats. More than 90% of the recorded neurons of both species had receptive fields (RFs) on single vibrissae. Movements of RF-vibrissae produced a burst of multiple discharges in S-TR neurons and single spike discharges followed by a prominent suppression of spontaneous discharges in VB neurons. Antidromic invasion from stimulation of the somatosensory cortex in VB neurons was suppressed after RF-vibrissae were stimulated. A possible functional organization comprising VB and S-TR neurons for processing impulses of vibrissal movements was suggested.

Principal component analysis of Motokawa's data on wavelength dependence of retinal processes.

Electric excitability of the human eye as determined by measuring the threshold for a sensation of phosphene in response to electric stimulation of the eye was found by Koiti Motokawa to increase temporarily after a brief illumination (J. Neurophysiol., 1949, 112, 475-488). While changing the wavelength of illuminating light widely, he found that the time course of the variation in the eye's electric excitability after illumination differed characteristically according to the wavelength. His data on this point (Tohoku J. exp. Med., 1949, 51, 197-205) were subjected to the principal component analysis. Three components were found necessary and sufficient for their linear combinations to reproduce time courses of the excitability enhancement after illumination with lights of varying wavelengths; one of the three components makes a great contribution to the excitability enhancement by green lights, the other to the one by red lights and the remainder to the one by blue lights. This is in support of Motokawa's view that his data are interpretable as summation effects of the three retinal processes which are excited preferentially by red, green and blue lights, respectively.

Effects of locus coeruleus stimulation on neuronal activities of dorsal lateral geniculate nucleus and perigeniculate reticular nucleus of the rat.

In rats anesthetized with urethane, a stimulating electrode was introduced to the locus coeruleus by observing the antidromic field response to single shock stimulation of the dorsal pathway of noradrenergic axons. Effects of locus coeruleus stimulation were studied on activities of relay neurons and intrinsic interneurons of the dorsal lateral geniculate nucleus and on those of neurons in the perigeniculate reticular nucleus. The intrinsic interneurons and the perigeniculate reticular neurons are believed to exert inhibition upon the relay neurons. The relay neurons were activated by repetitive stimulation of locus coeruleus; spontaneous discharges were increased in rate and the threshold of response to single shock stimulation of the optic nerve was lowered. The activation was rarely seen in rats pretreated with alpha-methyl-p-tyrosine. Iontophoretic application of phentolamine, an alpha-blocker, effectively antagonized the activation, whereas an iontophoretic beta-blocker and cholinergic blockers were virtually ineffective. The activation of the relay neurons was suggested to be due to a direct action of noradrenaline, released by locus coeruleus stimulation. Locus coeruleus stimulation inhibited the interneurons and activated the perigeniculate reticular neurons; spontaneous or light-evoked discharges were suppressed in the interneurons and tonic discharges were elicited in the perigeniculate reticular neurons. These effects of locus coeruleus stimulation were mimicked by noradrenaline applied iontophoretically. Activation of the perigeniculate reticular neurons was antagonized by an iontophoretic alpha-blocker but not by a beta-blocker. Two special features emerge from the present results: (1) the locus coeruleus exerts different effects upon the two neuronal constituents of the dorsal lateral geniculate nucleus, excitation of the relay neurons and inhibition of the intrinsic interneurons; (2) a suggestion previously advocated that locus-coeruleus-induced excitation of the lateral geniculate relay neurons would be due to inhibition of inhibitory neurons (disinhibition) does not hold true, at least with respect to the perigeniculate reticular neurons; the latter neurons have been proved to exert a powerful inhibition upon the geniculate relay neurons and they are excited by stimulation of the locus coeruleus.

Two pathways from the facial skin to the superior colliculus in the rat.

In response to single shock stimulation of the facial skin, the field potential of two negative waves (N1 and N2) appear in the intermediate layers of the rat superior colliculus (SC). From the experiments of functional ablations of the cortical facial area, the N1 wave was ascribed to the post-synaptic activity mediated by the direct trigemino-tectal pathway whereas the N2 wave to that mediated by the trigemino-cortico-tectal pathway. Single unit recordings also confirmed the existence of two pathways from the facial skin to the intermediate layers of SC.

Sensitivity to GABA of neurons of the dorsal and ventral lateral geniculate nuclei in the rat.

GABA was applied iontophoretically to dorsal and ventral lateral geniculate (LGd and LGv) neurons in rats. Spontaneous discharges were readily suppressed in both species of neurons. While in LGd neurons, evoked discharges by optic nerve stimulation were suppressed as readily as were spontaneous discharges, LGv neurons were characterized in that evoked discharges were much more resistant than spontaneous discharges.

Properties of ipsilateral retinogeniculate afferents in albino and hooded rats.

By recording single unit activities from the dorsal lateral geniculate nucleus in albino and hooded rats, physiological properties of the ipsilateral retinogeniculate afferents were compared with those of the contralateral ones. The results show that the ipsilateral retinogeniculate pathway was characterized by intermediate conduction velocities, relatively high incidence of the tonic response and the visual field representation of central 30 degrees from the vertical midline on both sides.

Inhibition of neuronal activity of the substantia nigra by noxious stimuli and its modification by the caudate nucleus.

In urethane-anesthetized rats discharges of neurons of substantia nigra, pars compacta (SNC) were recorded extracellularly after natural somatic sensory stimulation and electrical stimulation of peripheral sensory nerves. (1) Among different modalities of somatic sensory stimulation tested, noxious stimuli were effective in reducing spontaneous discharges of SNC neurons. The inhibition appeared with a concomitant increase of spike amplitude. The same inhibitory effect was obtained by stimulating the sciatic nerve (SC) repetitively. In response to single shock stimulation of the SC the inhibition occurred at an average latency of 39.6 msec (S.E. 1.6 msec) and lasted for 221.6 msec on average (S.E. 10.8 msec). (2) The SC-induced inhibition of SNC neurons failed to reliably block ortho- and antidromic discharges evoked from the caudate nucleus (Cd). (3) In rats with the Cd lesioned the SC-induced inhibition was longer lasting than in controls. When the Cd was stimulated concurrently with SC stimulation, the inhibition from the SC was weakened. (4) In a majority of SNC neurons, their inhibition by SC stimulation, their inhibition by SC stimulation was antagonized by intravenous injection of haloperidol.

GABA sensitivity of neurons of the visual layer in the rat superior colliculus.

Inhibitory action of iontophoretically applied GABA was examined on neurons in the visual layer of the rat superior colliculus (SC). Spontaneous discharges of all neurons tested were readily abolished by GABA ejected with currents less than 25 nA. In some cells the discharges evoked by near threshold electrical stimulation of the optic nerve or those evoked by a spot of light moving across receptive fields were suppressed by the same dose of GABA as that required to abolish the spontaneous discharge. However, in other cells the evoked discharges were much more resistant to GABA than the spontaneous activity. GABA sensitivity of the evoked activities was examined on various classes of SC cells which were identified by their recording depth, response latency to electrical stimulation of the optic chiasm and other properties. SC cells of the visual layer were classified into 8 types: classes Ia and Ib in the most superficial layer (N3 zone), class II in the thin layer below the N3 (N2 zone) and classes IIIa, IIIb, IVb and IVc in the deepest layer below the N2 (N1 zone). Effects of GABA upon these cell classes are summarized as follows; (1) Ia and IVb cells were readily suppressed by GABA, (2) Ib and II and most of IIIa and IVc cells were GABA-insensitive, and (3) GABA sensitivity varied from cell to cell in classes IIIb and IVa.