Long-term nitrogen compound removal trends of a hybrid subsurface constructed wetland treating milking parlor wastewater throughout its 7 years of operation.

This study evaluated the nitrogen compound removal efficiency of a hybrid subsurface constructed wetland, which began treating milking parlor wastewater in Hokkaido, northern Japan, in 2006. The wetland's overall removal rates of total nitrogen (TN) and ammonium (NH4(+)-N) improved after the second year of operation, and its rate of organic nitrogen (Org-N) removal was stable at 90% efficiency. Only nitrate (NO3(-)-N) levels were increased following the treatment. Despite increased NO3(-)-N (maximum of 3 mg-N/L) levels, TN removal rates were only slightly affected. Removal rates of TN and Org-N were highest in the first vertical bed. NH4(+)-N removal rates were highest in the second vertical bed, presumably due to water recirculation and pH adjustment. Concentrations of NO3(-)-N appeared when total carbon (TC) levels were low, which suggests that low TC prevented complete denitrification in the second vertical bed and the final horizontal bed. In practice, the beds removed more nitrogen than the amount theoretically removed by denitrification, as calculated by the amount of carbon removed from the system. This carbon-nitrogen imbalance may be due to other nitrogen transformation mechanisms, which require less carbon.

Performance of hybrid subsurface constructed wetland system for piggery wastewater treatment.

The objective of this study was to evaluate performance of a hybrid constructed wetland (CW) built for high organic content piggery wastewater treatment in a cold region. The system consists of four vertical and one horizontal flow subsurface CWs. The wetland was built in 2009 and water quality was monitored from the outset. Average purification efficiency of this system was 95±5, 91±7, 89±8, 70±10, 84±15, 90±6, 99±2, and 93±16% for biochemical oxygen demand (BOD5), chemical oxygen demand (COD), total carbon (TC), total nitrogen (TN), ammonium-N (NH4-N), total phosphorus (TP), total coliform (T. Coliform), and suspended solids (SS), respectively during August 2010-December 2013. Pollutant removal rate was 15±18 g m(-2) d(-1), 49±52 g m(-2) d(-1), 6±4 g m(-2) d(-1), 7±5 g m(-2) d(-1), and 1±1 g m(-2) d(-1) for BOD5, COD, TN, NH4-N, and TP, respectively. The removal efficiency of BOD5, COD, NH4-N, and SS improved yearly since the start of operation. With respect to removal of TN and TP, efficiency improved in the first three years but slightly declined in the fourth year. The system performed well during both warm and cold periods, but was more efficient in the warm period. The nitrate increase may be attributed to a low C/N ratio, due to limited availability of carbon required for denitrification.

Design and performance of hybrid constructed wetland systems for high-content wastewater treatment in the cold climate of Hokkaido, northern Japan.

The performance of six multistage hybrid constructed wetland systems was evaluated. The systems were designed to treat four kinds of high-content wastewater: dairy wastewater (three systems, average inflow content 2,400-5,000 mg·COD l(-1), 3-6 years of operation); pig farm wastewater, including liquid food washing wastewater (one system, 9,500 mg·COD l(-1), 3 years); potato starch processing wastewater (one system, 20,000-60,000 mg·COD l(-1), 3 years); and wastewater containing pig farm swine urine (one system, 6,600 mg·COD l(-1), 2.8 years) (COD = chemical oxygen demand). The systems contained three or four vertical (V) flow beds with self-priming siphons and surface partitions and no or one horizontal (H) flow bed (three to five beds). In some V flow beds, treated effluents were recirculated (Vr) through the inlet to improve performance. Mean annual temperature was 5-8 °C at all locations. To overcome clogging due to the high load in a cold climate, we applied a safety bypass structure and floating cover material to the V flow beds. Calculated average oxygen transfer rates (OTRs) increased proportionally with the influent load, and the OTR value was Vr > V> H. The relations of load-OTR, COD-ammonium, and a Arrhenius temperature-dependent equation enable the basic design of a reed bed system.

Erythroderma as a paraneoplastic cutaneous disorder in systemic anaplastic large cell lymphoma.

BACKGROUND: Paraneoplastic cutaneous disorders (PCDs) or dermadromes are skin conditions that have an association with internal malignancies but are not themselves malignant. We report the first two cases of systemic anaplastic large cell lymphoma (s-ALCL) accompanied by erythroderma and multiple leg ulcers as PCDs. CASE 1: A 52-year-old Japanese man presented with disseminated itchy papular erythemas which he had over his entire body for the preceding 5 years that later exacerbated to erythroderma. Multiple punched-out ulcers also developed on his lower legs. Superficial lymph nodes (LNs) were swollen, and a left axillary LN biopsy demonstrated dense CD30(+) atypical large cell (ALC) infiltration. By contrast, lymphocytes infiltrating into the erythroderma and leg ulcers were CD30(-) , and T-cell receptor ß (TCRß) chain gene rearrangement was negative in skin biopsy specimens. Thus, he was diagnosed with s-ALCL. Not only his s-ALCL but also his erythroderma and leg ulcers responded well to chemotherapy. CASE 2: A 71-year-old Japanese woman presented with erythroderma that persisted for approximately 20 years after mastectomy. At her initial hospital visit, she was diagnosed with s-ALCL by biopsy of swollen left inguinal LNs. Similar to Case 1, CD30(+) ALCs were negative in skin samples with normal TCRß chain gene rearrangement. As the erythrodermic skin lesion responded well to chemotherapy for s-ALCL, it was considered a PCD. CONCLUSION: s-ALCL development may be predicted by the precedence and concurrence of intractable paraneoplastic erythrodermic and ulcerative skin lesions, as reported in our two cases.

Expression of the 180-kD ribosome receptor induces membrane proliferation and increased secretory activity in yeast.

Expression of the canine 180-kD ribosome receptor (p180) in yeast cells resulted in a marked proliferation of intracellular membranes. The type of membranes observed varied with the expression of specific portions of p180. Rough membranes predominated when the ribosome binding domain of p180 was present, whereas expression constructs lacking this region resulted in smooth membranes. Northern analysis indicated that expression of the NH(2)-terminal 767 amino acids (DeltaCT), which include the ribosome binding domain, upregulated the transcription and translation of genes involved in exocytosis. The membranes that were proliferated were functional as these cells overcame a temperature-sensitive translocation defect. Most significantly, cells that overexpressed DeltaCT and proliferated rough endoplasmic reticulum exhibited severalfold higher levels of secretion of an ectopically expressed secretory protein. We conclude that p180 expression triggers a cascade of events leading to an increase in secretory potential akin to the terminal differentiation of mammalian secretory cells and tissues.

Cell Differentiation and Morphogenesis Are Uncoupled in Arabidopsis raspberry Embryos.

We identified two Arabidopsis embryo mutants, designated as raspberry1 and raspberry2, by screening T-DNA-mutagenized Arabidopsis lines. Embryogenesis in these mutants is indistinguishable from that of wild-type plants until the late-globular stage, after which raspberry1 and raspberry2 embryos fail to undergo the transition to heart stage, remain globular shaped, and proliferate an enlarged suspensor region. raspberry1 and raspberry2 embryo-proper regions enlarge during embryogenesis, become highly vacuolate, and display prominent convex, or "raspberry-like" protuberances on their outer cell layers. In situ hybridization studies with several embryo cell-specific mRNA probes indicated that the raspberry1 and raspberry2 embryo-proper regions differentiate tissue layers in their correct spatial contexts and that the regulation of cell-specific genes within these layers is normal. Surprisingly, a similar spatial and temporal pattern of mRNA accumulation occurs within the enlarged suspensor region of raspberry1 and raspberry2 embryos, suggesting that a defect in embryo-proper morphogenesis can cause the suspensor to take on an embryo-proper-like state and differentiate a radial tissue-type axis. We conclude that cell differentiation can occur in the absence of both organ formation and morphogenesis during plant embryogenesis and that interactions occur between the embryo-proper and suspensor regions.

LEAFY COTYLEDON1 Is an Essential Regulator of Late Embryogenesis and Cotyledon Identity in Arabidopsis.

LEAFY COTYLEDON1 (LEC1) is an embryo defective mutation that affects cotyledon identity in Arabidopsis. Mutant cotyledons possess trichomes that are normally a leaf trait in Arabidopsis, and the cellular organization of these organs is intermediate between that of cotyledons and leaves from wild-type plants. We present several lines of evidence that indicate that the control of late embryogenesis is compromised by the mutation. First, mutant embryos are desiccation intolerant, yet embryos can be rescued before they dry to yield homozygous recessive plants that produce defective embryos exclusively. Second, although many genes normally expressed during embryonic development are active in the mutant, at least one maturation phase-specific gene is not activated. Third, the shoot apical meristem is activated precociously in mutant embryos. Fourth, in mutant embryos, several genes characteristic of postgerminative development are expressed at levels typical of wild-type seedlings rather than embryos. We conclude that postgerminative development is initiated prematurely and that embryonic and postgerminative programs operate simultaneously in mutant embryos. The pleiotropic effects of the mutation indicate that the LEC1 gene plays a fundamental role in regulating late embryogenesis. The role of LEC1 and its relationship to other genes involved in controlling late embryonic development are discussed.

Cell cycle parameters of Chinese hamster ovary cells during exponential, polyamine-limited growth.

We have previously shown that Chinese hamster ovary cells made polyamine deficient by treatment with alpha-methylornithine, an inhibitor of ornithine decarboxylase, grow exponentially in culture at low densities at one-half the rate observed in untreated (control) cultures. In this study, the cell cycle of polyamine-limited cells was examined by using thymidine autoradiography, mitotic index analysis, and fraction labeled mitoses analysis. We found that the longer doubling time of inhibitor-treated cultures was a consequence of increases in the lengths of the G1 and S phases. The expansion of the S phase was proportional to the increase in doubling time (twofold), whereas the G1 phase was lengthened by slightly more than a factor of 2. The lengths of the G2 and M phases were essentially unchanged. Putrescine stimulated the growth of inhibitor-treated cultures and restored the cell cycle parameters to those of untreated cells.

Signaling in plant embryogenesis.

Embryogenesis is a critical stage of the sporophytic life cycle during which the basic body plan of the plant is established. Although positional information is implicated to play a major role in determining embryo cell fate, little is known about the nature of positional signals. Recent studies show that the monopterous and hobbit mutations reveal signaling during patterning of the embryonic axis. The LEAFY COTYLEDON1 and PICKLE genes have been implicated to play important roles in controlling embryo development.

Evidence for receptor-mediated inhibition of intrinsic activity of GTP-binding protein, Gi1 and Gi2, but not G0 in reconstitution experiments.

The receptor-mediated inhibition of intrinsic activities of GTP-binding proteins (G-proteins) was studied. Pertussis toxin (IAP)-substrate G-protein, Gi1, Gi2 or G0, was prelabeled with [alpha-32P]GDP and reconstituted with synaptic membranes of the guinea pig cerebellum in the presence of 0.02% of Chaps. Intrinsic activities of G-proteins were evaluated by the release of [alpha-32P]GDP in exchange for added GppNHp or GDP in reconstituted preparations. U-50,488H (1 nM-10 microM), a specific kappa-subtype of opioid receptor agonist, inhibited the [alpha-32P]GDP release in exchange for added 1 microM GppNHp in Gi1-reconstituted preparations in a concentration-dependent manner. On the other hand, the kappa-opioid agonist at 10 microM increases the Km values of GppNHp, but not GDP in exchange for [alpha-32P]GDP release in preparations reconstituted with Gi1 or Gi2, but not with G0. These findings indicate that kappa-opioid receptor is coupled to inhibition of intrinsic activities of Gi1 and Gi2, but not G0, in guinea pig cerebellar membranes. In addition, it was revealed that the mode of action is mediated by a decrease in affinity of GTP (or its analog) for G proteins, but not by a change in affinity of GDP.

Tumor DNA content in primary and metastatic colorectal carcinoma.

We performed this study to determine if tumor DNA content was an independent prognostic indicator in patients with primary tumors or hepatic metastases from colorectal cancer. We analyzed tumor DNA content by flow cytometry from paraffin-embedded specimens in 133 patients. In the 77 patients with primary colorectal cancer who had had "curative" resection, DNA content of the tumor was an independent prognostic indicator in predicting both relapse-free and overall survival. Logistics regression analysis demonstrated that aneuploidy, ie, tumors that exhibited a population of cells with an "abnormal" DNA content, was the single most important variable in predicting both recurrence and death from disease, even more important than stage. However, in 56 patients with hepatic metastases from colorectal cancer, the DNA content of the liver metastases bore no relationship to the patients' survival or their response to therapy. These data suggest that tumor DNA content is an important prognostic indicator in patients with primary colorectal carcinoma but does not reflect the natural history of patients with liver metastases.

Activation of caspase-3 in beta-amyloid-induced apoptosis of cultured rat cortical neurons.

Amyloid beta protein (Abeta) has been thought to participate in the neurodegeneration associated with Alzheimer's disease. We here report on caspase-3 activation by Abeta-treatment of cultured neurons. Treatment of rat primary cortical culture with Abeta 25-35, an active fragment of Abeta, induced neuronal death as determined by a decrease in neuron-specific microtubule-associated protein 2 (MAP2)-like immunoreactivity and by the release of cellular lactate dehydrogenase (LDH). Abeta 25-35 also induced elevation of caspase-3-like Ac-DEVD-MCA cleavage activity in advance of neuronal death with similar concentration-dependency for neuronal death. Inhibitor sensitivity of the Abeta-induced proteolytic activity was similar to that of human recombinant caspase-3. Cleavage of pro-caspase-3 and cleavage of its endogenous substrates, poly (ADP-ribose) polymerase (PARP) and alpha-fodrin, were produced by Abeta-treatment. A caspase-3 inhibitor, Ac-DEVD-CHO, prevented Abeta-induced DNA fragmentation and cleavage of alpha-fodrin, but not of PARP. Caspase inhibitor of broad specificity, Z-VAD-CH(2)-DCB, additionally prevented Abeta-induced cleavage of PARP and some early loss of cell membrane integrity measured by LDH release. However, Abeta-induced condensation of nuclear chromatin and most of the late disintegration of cell membranes were not prevented in the presence of these caspase inhibitors. These results suggest that activation of both caspase-3 and caspase(s) other than caspase-3 play distinct roles in Abeta-induced apoptosis of rat cortical neurons. Furthermore, in the presence of caspase inhibitors, Abeta-induced neuronal death still occurred with different morphological features.

Inhibitors of interleukin-1 beta-converting enzyme-family proteases (caspases) prevent apoptosis without affecting decreased cellular ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide in cerebellar granule neurons.

We assessed the possible role of interleukin-1beta-converting enzyme-family proteases (caspases) in apoptosis in cultured rat cerebellar granule neurons. CPP32 (caspase-3)-like protease activity was augmented by low KCl treatment, preceding neuronal cell death. Agents such as brain-derived neurotrophic factor (BDNF), dibutylyl cAMP, NMDA, actinomycin D, S-adenosyl-L-methionine, and spermine prevented apoptosis. For various neuroprotective agents, the degree of apoptosis prevention correlated with the prevention of the activation of CPP32-like protease. Furthermore, Z-Asp-2, 6-dichlorobenzoyloxy-methylketone (Z-Asp-CH2-DCB), Boc-Asp-fluoromethylketone (Boc-Asp-FMK), and Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), which are inhibitors of caspases, also prevented apoptosis. In contrast to many other neuroprotective agents, these inhibitors of caspases showed little effect on the decrease of cellular 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) reduction activity after low KCl treatment. The neurons rescued by these inhibitors of caspases during low KCl treatment were in a hypoenergic state in their ATP levels and vulnerable to subsequent treatment with medium containing high KCl or glutamate which induce an influx of Ca2+, but which are less toxic to normal neurons. These results suggest that caspase(s) are involved in the apoptosis of cerebellar granule neurons and that several agents protect neurons from death by blocking the activation of the protease(s). Although several caspase inhibitors examined in this study protect neurons from apoptosis, rescued neurons are vulnerable to subsequent stimuli that induce necrotic cell death.

Polyamines prevent apoptotic cell death in cultured cerebellar granule neurons.

Polyamines play critical roles during the development of brain neurons. In the present study we examined the effects of polyamines on neuronal apoptotic death. Rat cerebellar granule neurons were cultured in the presence of a depolarizing concentration of KCl (25 mM) in the medium. Apoptotic neuronal death was induced by changing the medium to that containing 5.6 mM KCl without serum. Spermine as well as spermidine and putrescine prevented cell death in a concentration-dependent manner with the order of potency being spermine > spermidine > putrescine. The effect of spermine was partially blocked by several NMDA-type glutamate receptor antagonists including (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801). MK-801-sensitive neuroprotection by spermine depended on cell density. Activation of CPP32 (caspase-3/Yama/apopain)-like proteolytic activity, a key mediator of apoptosis, precedes neuronal death, and polyamines prevented an increase in this activity. These results demonstrate that polyamines protect neurons from apoptotic cell death through both NMDA receptor-dependent and -independent mechanisms, acting upstream from the activation of CPP32-like protease(s).

Affinity cross-linked delta-opioid receptor in NG108-15 cells is low molecular weight (25 kDa) and coupled to GTP-binding proteins.

The affinity cross-linking of the delta-opioid receptor in neuroblastoma x glioma NG108-15 cells was undertaken using (3-[125I]iodotyrosyl27)human-beta-endorphin ([125I]beta-endorphin) and disuccinimidyl suberate (DSS) or bis(sulfosuccinimidyl) suberate (BS3) in order to estimate molecular size. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, two radioactive bands were observed. Labeling of a major band of 29 kDa diminished in the presence of unlabeled selective delta-opioid agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), in a concentration-dependent manner, while labeling of a minor band of 58 kDa was hardly affected. The labeling intensity of the 29 kDa band decreased by addition of guanosine 5'-(3-o-thio)triphosphate (GTP gamma S) or by pretreatment of cells with pertussis toxin. These results, taking the molecular weight of covalently bound beta-endorphin (3.6 kDa) into consideration, suggest that the delta-opioid receptor in NG108-15 cell membrane is a 25 kDa protein which is coupled to pertussis toxin-sensitive guanosine triphosphate-binding proteins (G-proteins).

Allelochemicals of the tropical weed Sphenoclea zeylanica.

Nine plant growth inhibitors were isolated from the tropical weed Sphenoclea zeylanica, which shows allelopathic properties. Those compounds hitherto not reported from any plant source were the isomers of cyclic thiosulfinate, (1S,3R,4R)-(+)- and (1R,3R,4R)-(+)-4-hydroxy-3-hydroxymethyl-1,2-dithiolane-1-oxides, and (2R,3R,4R)-(-)- and (2S,3R,4R)-(+)-4-hydroxy-3-hydroxymethyl-1,2-dithiolane-2-oxides. These were named zeylanoxide A, epi-zeylanoxide A, zeylanoxide B and epi-zeylanoxide B, respectively. The absolute configurations at C-3 and C-4 were elucidated by chemical synthesis of both enantiomers from L- and D-glucose. Two of the inhibitors were secologanic acid and secologanoside. and three other inhibitors were by known secoiridoid glucosides formed as artifacts during extraction with methanol. The cyclic thiosulfinates and secoiridoid glucosides completely inhibit the root growth of rice seedlings at 3.0 mM. While the specific activity of the inhibitors was not high, since they accumulated to circa 0.61% S. zelanica by dry weight, this suggests that the inhibitors are nervertheless potent allelochemicals in this weed.

The characteristics of laser-Doppler flowmetry for the measurement of regional cerebral blood flow.

The fundamental characteristics of laser-Doppler flowmetry (LDF), especially the depth of cerebral blood flow (CBF) measurement, have not been widely studied in the brain tissue; however, LDF has been widely used in recent clinical and experimental studies. We investigated the depth of CBF measurement and other characteristics related to the use of LDF in the brain. In an animal experimental study, the distribution of laser light and the depth of CBF measurement of LDF were measured by using modified LDF probes. CBF in various conditions was also measured by the LDF and hydrogen clearance method. Laser light of low output lost directivity and was dispersed into a hemispherical form in the brain tissue. The depth of CBF measurement was approximately 100 to 400 microns, depending on the intensity of the emitted laser light, and was affected by changes of CBF. In the physiological condition, the close correlation between the values of CBF by the LDF and hydrogen clearance method was obtained. After cardiac arrest, the CBF value of LDF did not immediately show a 0 value. LDF has several special characteristics, and the sample volume was very small. It is important to pay attention to the several special characteristics of LDF.

Modified thermal diffusion flow probe for the continuous monitoring of cortical blood flow.

A small thermal diffusion flow probe has been developed to monitor the dynamic changes in cerebral blood flow in small animals. Constantan wire was used as a heat source to make a miniature probe. The pair of thermocouples used to detect the heat gradient between two gold plates was elongated to avoid heat conduction between them, and this improvement allowed us to make quantitative measurements. After several basic experiments, local cerebral blood flow was measured simultaneously, using both the modified thermal probe and the hydrogen clearance method in four rabbits. A close relationship was obtained between the local cerebral blood flow values measured by hydrogen clearance (F, ml/100g/min) and the reciprocal of the thermocouple voltage (1/V;1/mV). The regression line was F = 29111(1/V - 1/226), (r = 0.92, P less than 0.001). We suggest that the modified thermal probe is a reliable and quantitative means of measuring flow. In addition, another probe modified for clinical use was evaluated. Continuous monitoring of local cerebral blood flow in postoperative patients was performed, and some illustrative cases are described.

Differences in critical cerebral blood flow with age in swine.

Normal cerebral blood flow (CBF), critical CBF at a flat reading of the electroencephalogram (EEG), and reversibility of the flat EEG after reperfusion were investigated in a total of 59 pigs, including seven newborns (1 to 3 days of age), 38 juveniles (1 month old), and 14 adults (7 months old). The CBF was determined by the hydrogen clearance method; the EEG was recorded continuously and a power spectrum analysis was performed. Cerebral ischemia was produced by occlusion of both common carotid arteries and induction of hypotension (approximately 50 mm Hg). The flat EEG reversibility was investigated for 3 hours after reperfusion. As parameters of brain development, the neuronal density and the time at which the S-100 protein appeared in the brain were examined. Normal CBF was highest in neonatal pigs and decreased with age. The critical CBF at a flat EEG was lowest in newborn pigs and was elevated with development of the brain. Tolerance against cerebral ischemia was greatest in newborn pigs.

Percutaneous needle muscle biopsy in the diagnosis of neuromuscular disorders in children. Histological, histochemical and electron microscopic studies.

Percutaneous needle biopsy was carried out in 75 patients with various neurological and muscle diseases (myopathies in 7, lower motor neuron diseases in 22, upper motor neuron diseases in 6, and unlocalized or miscellaneous diseases in 13) and its usefulness as a diagnostic procedure was evaluated. The ages of the subjects ranged from 2 months to 35 years, 77% were younger than 6 years. The materials were examined histologically, histochemically and electron microscopically with the following results. 1. The muscle volume obtained by needle biopsy was sufficient for studies in 64 cases (85%), insufficient in 8 (11%), while in 3 cases no muscle tissue was obtained. 2. Histological staining properties were excellent in all samples examined. In histochemical preparations, staining was judged as good in 61 cases, but poor in 11 due to an inappropriate freezing procedure. 3. In 33 cases, both needle and open biopsies were carried out simultaneously, and the morphological findings of the specimens were compared. There were no significant differences between them histologically as well as histochemically. Electron microscopic examinations also provided identical information in the seven cases studied so far. 4. The method of needle biopsy has many advantages over open biopsy as a routine diagnostic procedure for neuromuscular disorders in infants and young children, but there are a few disadvantages such as the requirement of technical skill and the inappropriateness for histogram construction in some cases.