Effect of osmolarity on glycosaminoglycan production and cell metabolism of articular chondrocyte under three-dimensional culture system.

OBJECTIVES: This study examined how physiological levels of extracellular osmolarity influence proteoglycan accumulation in articular chondrocytes in a three-dimensional culture system. METHODS: Cells were obtained from metacarpal phalangeal joints of 18-24 month bovine. They were cultured for 6 days in alginate beads at 4 million cells/ml in DMEM containing 6% FBS under 21% O2. Medium osmolarity was altered by NaCl addition over the range 270-570 mOsm and monitored using a freezing point osmometer. Profiles across intact beads were determined by manual counting using fluorescent probes and transmission electron microscope. Lactate production was measured enzymatically and glycosaminoglycan (GAG) accumulation was measured using a modified dimethylmethylene blue assay. Rate of sulfate GAG synthesis was measured using a standard 35S-sulfate radioactive method. RESULTS: The cell viability was similar for the high and low osmolarity cultures. However, confocal microscopy showed that the cells were the largest under 270 mOsm and became smaller with increasing osmotic pressure. GAG production was largest in the 370mOsm, and the capacity for GAG production and cell metabolism (lactate production) was low under hypo-osmolarity and hyper-osmolarity, and cell deaths were often observed on electron microscopy. CONCLUSIONS: In our model the prevailing osmolarity was a powerful regulator of GAG accumulation by cultured chondrocytes. These results thus indicate GAG synthesis rates are regulated by GAG concentration, with implications both for the aetiology of osteoarthritis and for tissue engineering.

Imaging of cauda equina edema in lumbar canal stenosis by using gadolinium-enhanced MR imaging: experimental constriction injury.

BACKGROUND AND PURPOSE: It has been reported that disturbance of blood flow arising from circumferential compression of the cauda equina by surrounding tissue plays a major role in the appearance of neurogenic intermittent claudication (NIC) associated with lumbar spinal canal stenosis (LSCS). We created a model of LSCS to clarify the mechanism of enhancement within the cauda equina on gadolinium-enhanced MR images from patients with LSCS. METHODS: In 20 dogs, a lumbar laminectomy was performed by applying circumferential constriction to the cauda equina by using a silicon tube, to produce 30% stenosis of the circumferential diameter of the dural tube. After 1 and 3 weeks, gadolinium and Evans blue albumin were injected intravenously at the same time. The sections were used to investigate the status of the blood-nerve barrier function under a fluorescence microscope and we compared gadolinium-enhanced MR images with Evans blue albumin distribution in the nerve. The other sections were used for light and transmission electron microscopic study. RESULTS: In this model, histologic examination showed congestion and dilation in many of the intraradicular veins, as well as inflammatory cell infiltration. The intraradicular edema caused by venous congestion and Wallerian degeneration can also occur at sites that are not subject to mechanical compression. Enhanced MR imaging showed enhancement of the cauda equina at the stenosed region, demonstrating the presence of edema. CONCLUSION: Gadolinium-enhanced MR imaging may be a useful tool for the diagnosis of microcirculatory disorders of the cauda equina associated with LSCS.

Co-digestion of domestic kitchen waste and night soil sludge in a full-scale sludge treatment plant.

A study was made on the domestic kitchen waste and night soil treatment performance of a full-scale sludge treatment plant. The sludge treatment at this plant was by thermophilic methane fermentation. The initial treatment, mesophilic to thermophilic fermentation, was able to be started up within a short time by adjusting the amount of influent waste. Thermophilic methane fermentation was carried out for five months (May-October) and the performance under a mean residual time of 22 days indicated a VTS decomposition of 42%, gas generation of 54-1,610 m3/day (average: 755 m3/day), and a mean methane concentration of 60%. The methane gas was used to generate power in the plant and the amount of power generated by methane gas was highest in October (average of 1,200 kWh/day). This was equivalent to about 7% of the power consumed at the entire sludge treatment plant. The BOD/NH4-N of the activated sludge influent water was lower, compared to a case where there is no recycle flow, due to the recycle flow from the methane fermentation process. There was, therefore, a tendency for an increase in the amount of methanol charged into the secondary denitrification tank. However, the quality of the effluent was satisfactory (BOD< 10 mg/L, SS< 5 mg/L, and T-N< 25 mg/L). Study results indicated that it was possible to implement a full-scale plant for recovering organic waste.

Methane fermentation of coastal mud sediment by a two-stage upflow anaerobic sludge blanket (UASB) reactor system.

The removal of organic matter from a coastal mud sediment was carried out by a methane fermentation process under anaerobic conditions. In a batch acidogenic fermentation, the addition of vitamins containing thiamine, nicotinic acid and biotin dramatically enhanced acetate production from the mud sediment (200 g wet wt l(-1) artificial sea water), yielding 77 mM acetate after 6 days, which corresponded to 77% of the organic matter in the mud sediment, measured on the basis of chemical oxygen demand. Thereafter, the two-fold diluted, post-acidogenic fermentation liquor (PAF liquor) was continuously treated at 2.4x original dilution rate day(-1) for 30 days, using an upflow anaerobic sludge blanket methanogenic reactor containing the acclimated methanogenic sludge from the mud sediment. Acetate, 42 mM in the PAF liquor, was converted to methane at a maximum methane production rate of 96 mmol l(-1) day(-1); and 87.5% of the acetate and 88.7% of the total organic carbon in the PAF liquor were removed. Moreover, an efficient treatment of the mud sediment was carried out by a semi-continuous, two-stage reactor system, where the culture broth was circulated between acidogenic and methanogenic reactors. This two-stage reactor system gave a stable operation at 4-day intervals for one treatment period, yielding 112 mmol methane from the wet mud in the PAF liquor (278 g l(-1)).

Treatment of aquarium water by denitrifying photosynthetic bacteria using immobilized polyvinyl alcohol beads.

During the purification of an aquarium for carp breeding, a relatively high level of chemical oxygen demand (COD) was removed by filtration systems packed with both alginate- and polyvinyl alcohol (PVA)-immobilized gel beads of Rhodobacter sphaeroides S. Low nitrate accumulation was observed in the alginate gel beads packed system due to denitrification, but high levels of nitrate and nitrite accumulation were observed in the PVA gel beads packed system. This phenomenon was caused by the inhibitory effect of PVA on nitrite reductase. The boric acid used for hardening gel beads of PVA slightly inhibited nitrate reductase. On the other hand, during the denitrifying growth experiments for this strain, boric acid inhibited cell growth, but PVA only partially inhibited cell growth. Based on electron equivalent (Y(eq.)), growth yields using various kinds of concentrations of PVA were almost identical. It was suggested that PVA might only limit the growth rate of this strain by the inhibition of nitrite reductase.

Removal of phosphorus from oyster farm mud sediment using a photosynthetic bacterium, Rhodobacter sphaeroides IL106.

Removal of phosphorous compounds from the mud sediment of an oyster farm was carried out by a series of bio-processes under anaerobic conditions. Anaerobic acidogenic fermentation of a mud sediment suspension (200 g wet wt/l artificial sea water) was initially carried out. With the addition of vitamins such as thiamine, nicotinic acid and biotin, acidogenic fermentation was enhanced to yield acetic acid of approximately 2 g/l. Furthermore, approximately 20 mg/l of PO4(3-) (10% of total phosphorus on mud weight) and 5300 mg/l of COD(Cr) (82% of organic matter on mud weight) were released into the culture broth after fermentation for 7 d. The supernatant of this culture broth was used to cultivate Rhodobacter sphaeroides IL106, a denitrifying photosynthetic bacterium. After 4 d, 3.32 g/l of biomass containing carotenoid and ubiquinone was obtained, and COD(Cr) and acetic acid were reduced by 58% and 72%, respectively. In addition, PO4(3-) was reduced by 97%, suggesting that the removal of PO4(3-) from the mud sediment might be possible by combining anaerobic acidogenic fermentation with R. sphaeroides cultivation.

Effects of systemic administration of 2-(4-phenyl-piperidino)-cyclohexanol (vesamicol) and an organophosphate DDVP on the cholinergic system in brain regions of rats.

Vesamicol is known to inhibit the transport of acetylcholine (ACh) into synaptic vesicles in vitro, but much less is known about its effects in the brain in vivo. To assess the effect of vesamicol in vivo, we examined cholinergic parameters, such as the subcellular distribution of ACh, activities of enzymes, uptake of choline, and muscarinic receptor binding in the striatum, hippocampus, and cerebral cortex of rats 30 and 60 min after intraperitoneal injection of vesamicol (3 mg/kg) or of vesamicol in combination with DDVP (5 mg/kg), which was administered 10 min before vasamicol. The levels of cytosolic ACh increased in all regions of the brain after injection of vesamicol, while those of vesicular ACh decreased in all regions except for the striatum. The increase in the levels of extracellular ACh and cytosolic ACh in the striatum induced by DDVP was generally enhanced after injection of vesamicol, Vesamicol did not reduce the level of vesicular ACh when DDVP had been injected previously. Vesamicol did not induce any significant changes in the activities of enzymes, choline uptake, or binding of [6H]quinuclidinyl benzilate to the muscarinic ACh receptors in the three regions. Changes in the cholinergic parameters caused by DDVP were not reversed by the combined administration of DDVP with vesamicol. The present results indicate that vesamicol can inhibit the transport of ACh into synaptic vesicles in the brain tissue in vivo, although it cannot reverse the effects of DDVP that has been injected prior to vesamicol.

Antioxidant action of thiopalmitic acid on microsomal lipid peroxidation.

The antioxidant action of thiopalmitic acid (SH-Pal) was studied in a lipid peroxidation system using microsomes from rat liver. The Fe(II)/ascorbic acid (AsA)-induced lipid peroxidation, as measured by the amount of thiobarbituric acid-reactive substances (TBA-RS), was progressively inhibited by the addition of increasing amounts of SH-Pal. The inhibitory effect of SH-Pal in this experimental system was greater than that of alpha-tocopherol, glutathione (GSH) and palmitic acid. The antioxidative effect was abolished gradually by the addition of increasing amounts of N-ethylmaleimide to the system. Similarly, microsomal lipid peroxidation induced by Fe(III)-ADP/NADPH or CCl4/NADPH was inhibited in a dose-dependent fashion by the addition of SH-Pal. Moreover, SH-Pal was able to reduce 1,1-diphenyl-2-picrylhydrazyl (DPPH). The alpha-tocopherol content of the microsomal lipid peroxidation system decreased rapidly when no SH-Pal was present. However, upon adding SH-Pal (90 microM), the decrease in the alpha-tocopherol content of the assay system was markedly reduced. These findings indicate that SH-Pal acts as an antioxidant and free radical scavenger in lipid peroxidation carried out by rat liver microsomes.

Characteristics of histamine release from rat mast cells induced by a bracken toxin, braxin A1.

The effect of braxin A1, a new bracken glucoside, on histamine release from isolated rat peritoneal mast cells was studied. Braxin A1 caused the release of histamine in a dose-dependent manner; the release was slow and increased gradually with time, finally reaching a maximum release of 100%. The action of braxin A1 depended on the incubation temperature in the range from 4 degrees C to 49 degrees C, while it was almost abolished at 0 degree C. The action of braxin A1 was unaffected by removing calcium or any inorganic ions from the incubation medium and by the addition of 2,4-dinitrophenol or theophylline. The mast cells exposed to braxin A1 were vitally stained with trypan blue and swelled greatly. The cell swelling was characterized by the protrusion of swollen cytoplasmic granules. The present results for braxin A1 were similar to those for the ionophore X537A except for the extracellular inorganic ion dependency, but they were different from those observed with compound 48/80. These results suggest that braxin A1 releases histamine from mast cells without both exocytosis and membrane lysis, but with a cytotoxic action on cytoplasmic membranes by a different mode of action from that of X537A.

Human pancreatic polypeptide responsiveness to insulin-induced hypoglycemia in anorexia nervosa.

Patients with anorexia nervosa occasionally suffer from hypoglycemic comas. We investigated the role of human pancreatic polypeptide (HPP) in insulin-induced hypoglycemia (0.1 U/kg of regular insulin). Ten female patients with anorexia nervosa (20.7 +/- 2.0 years, mean +/- SEM; 34.9 +/- 1.7 kg, mean +/- SEM) and 8 age-matched female controls (20.9 +/- 0.6 years, 51.5 +/- 0.8 kg) were tested. In the patients with anorexia nervosa, testing was performed before and after the restoration of body weight (45.0 +/- 0.8 kg). There was no significant difference in glucose nadir between patients with anorexia nervosa and the control subjects. However, glucose recovery from nadir was delayed in patients with anorexia nervosa. In anorexia nervosa patients, the plasma pancreatic glucagon responses to insulin-induced hypoglycemia did not differ from those of the controls. Results also showed, however, that HPP responses to insulin-induced hypoglycemia were significantly higher in patients with anorexia nervosa than in controls (p less than 0.01). The increased HPP responses were still present after the restoration of body weight in anorexia nervosa patients. A complete body weight recovery or a longer period of time may be required to normalize the HPP response to insulin-induced hypoglycemia in patients with anorexia nervosa, after the restoration of body weight.

[Microorganisms isolated from urinary tract infection and their beta-lactamase production and evaluation of clinical efficacy of sulperazone].

Clinical survey of microorganisms isolated from urinary tract infection (UTI) was carried out at the four major hospitals in Mie Prefecture from May to July, 1987, and production of beta-lactamase of the microorganisms was determined by the acidimetric method, "beta-checker". Among the total of 460 strains isolated from urine samples, 135 of gram positive cocci and 325 of gram negative rods were contained. Sixty percent of the gram negative rods and 14% of gram positive cocci produced beta-lactamase. Pseudomonas aeruginosa, E. coli and Serratia marcescens were representative organisms which produced beta-lactamase. Acinetobacter, Klebsiella, Enterobacter and Citrobacter species produced beta-lactamase at a higher rate but those were not so frequently isolated. In the sensitivity test to Sulperazone, the representative organisms isolated from urine, as a whole, had a sensitivity of 74% and E. coli, Klebsiella, S. epidermidis and Pseudomonas aeruginosa were highly sensitive, while Enterobacter cloacae and Serratia marcescens showed low sensitivity. The clinical efficacy of Sulperazone was evaluated in 26 patients with complicated UTI. Overall effectiveness rate and eradication rate of beta-lactamase production organisms were 73.1 and 63%, respectively. Sulperazone is concluded to be a useful antibiotic for treating complicated UTI induced by beta-lactamase production organisms from the point of microbiology and safety.

[Studies on histamine containing cells in the spleen of the magnesium-deficient rats].

When young Wistar rats (body wt. 50 g) were maintained on a magnesium-deficient diet (0.001% Mg) for eight days, the splenic weight and histamine content increased about 2-fold and 30-fold, respectively, compared with those of the control rats. There was no significant difference in the number of splenic mast cells between the magnesium-deficient and control rats. More neutrophilic, eosinophilic and basophilic granular cells were found in the spleen cells isolated from the magnesium-deficient rats than in those from the control rats. Of the isolated cells from magnesium-deficient rats, 7.6% were basophilic granular cells; however, no basophilic granular cells were observed in the spleen cells isolated from the control rats. In the cytochemical study, the yellowish fluorochrome formed by the interaction of o-phthalaldehyde and histamine was found in basophilic granular cells. These results suggest that the increase in the histamine content of the spleen of magnesium-deficient rats is related to the increased number of basophilic granular cells.

[Effects of magnesium deficiency on dermal mast cells in rats].

The effects of dietary magnesium (Mg) deficiency on dermal mast cells were studied in young Wistar rats weighing about 50 g. The rats fed with a Mg-deficient diet (0.001% Mg) showed hyperemia on the 3rd or the 4th day after they were fed the diet. The dermal mast cells of the control rats were filled with granules, while the cells of rats fed the Mg-deficient diet for 4 days contained less granules than the controls, but contained extensively dilated rough surfaced endoplasmic reticulum, well-developed Golgi complexes, many mitochondria and ribosomes. These data suggest that hypomagnesemia could induce a release of histamine from dermal mast cells. So, the effect of a low Mg medium on the release of histamine was studied using peritoneal mast cells in vitro. A low Mg medium (0.2 mM Mg) induced much more histamine release than control medium (1 mM Mg) from the peritoneal mast cells obtained from both control and Mg-deficient rats fed with the Mg-deficient diet for 2 days. The peritoneal mast cells obtained on the 8th day of Mg-deficiency released much more histamine than controls in 1 mM Mg medium. These results suggest that hyperemia observed in Mg-deficient rats depends partly on histamine released from dermal mast cells.

Abnormal serum prolactin responses to luteinizing hormone-releasing hormone (LHRH) in patients with anorexia nervosa and bulimia.

Abnormal responses of serum prolactin (PRL) to luteinizing hormone-releasing hormone (LHRH) stimulation have been observed in anovulatory women and in hypogonadal patients. Various endocrinological abnormalities have been demonstrated in patients with anorexia nervosa (AN). The present study was undertaken to further investigate responses of serum PRL, growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) to LHRH stimulation in 65 patients with AN and in 12 patients with bulimia before therapy and in the AN patients after several months of treatment, and in comparison to 12 normal women of the same age. Serum PRL responses to LHRH were positive (peak PRL levels greater than 25 ng/ml and delta increase in PRL greater than 10 ng/ml) in 16.9% of AN and 16.6% of bulimic patients; they were negative (absent) in all controls. Following restoration of the AN patients to normal body weight, the PRL responses to LHRH became normalized in those patients whose eating disorder behavior also returned to normal. However, in those patients whose eating disorder patterns continued to be abnormal, abnormal PRL responses persisted. The bulimic patients were of normal body weight, and yet had abnormal PRL responses. Thus, the responses of PRL correlated more closely with the behavior of the underlying eating disorder rather than with body weight gain or normal body weight.

Effect of psychological stress on human growth hormone response to thyrotropin-releasing hormone in normal controls.

Human growth hormone (hGH) responses to thyrotropin-releasing hormone (TRH) were investigated in normal subjects under psychological stress. Fifteen subjects (4 men and 11 women), whose ages ranged from 19 to 22 years, were studied. The mirror-drawing test (MDT) was performed to induce psychological stress. Plasma hGH and prolactin (PRL) were determined serially before, during and after the following tests: TRH alone (500 micrograms synthetic TRH i.v. bolus), MDT alone, and TRH with MDT. The changes in hGH concentrations with MDT alone were not significant. The hGH response to TRH alone also showed no remarkable change; however, hGH responses to TRH combined with MDT were significantly higher than the responses to TRH alone. PRL did not respond with MDT alone but responded significantly with the other two tests. Thus there was a divergence in hGH and PRL secretion to TRH and psychological stress. Significant increases in hGH secretion were observed only when TRH and psychological stress were combined as stimuli.

Involvement of the cholinergic mechanism in DDT and dieldrin action in the insect central nervous system.

The effects of DDT and dieldrin on cholinergic neurotransmission were studied using the sixth abdominal ganglion of the cockroach. Spontaneous electrical discharges in the ganglion recorded with an extracellular electrode were augmented by 0.1 mM DDT and 1 microM dieldrin. This stimulating action was partly blocked by 0.5 mM d-tubocurarine and 0.1 mM hemicholinium-3 and disappeared in a high Mg2+-low Ca2+ medium. DDT and dieldrin increased both ACh release and ACh content in the ganglion. These results suggested that DDT and dieldrin stimulate both ACh release and synthesis.

Involvement of cholinergic mechanism in the action of allethrin in insect CNS.

Allethrin had a stimulating action on spontaneous discharges in the cockroach sixth abdominal ganglion superfused with an insect saline solution. This action at a low concentration (5 X 10(-8) M) of allethrin was abolished by either of d-tubocurarine, hexamethonium or atropine at 5 X 10(-4) M. It was also abolished by the treatment of ganglia with hemicholinium-3 or by low-calcium-high magnesium insect saline solution. However, treatment of these blocked ganglia with allethrin at more than 5 X 10(-7) M overcame the block, producing increased spontaneous activity. Allethrin had no effect on insect cholinesterase activity. These results may suggest that the stimulating action at a low concentration of allethrin may be mediated by the release of ACh from cholinergic terminals in ganglia.

Endogenous Gibberellin-Like Substances in Somatic Embryos of Grape (Vitis vinifera x Vitis rupestris) in Relation to Embryogenesis and the Chilling Requirement for Subsequent Development of Mature Embryos.

Endogenous gibberellin (GA)-like substances were examined in suspension cultures of somatic embryos of a hybrid grape (Vitis vinifera x Vitis rupestris) during embryogenesis, and in mature embryos chilled at 4 degrees C, and subsequently incubated at 26 degrees C with and without abscisic acid (ABA). The extract was separated into a nonpolar fraction (would contain GA-precursors); a fraction that would contain free GAs; and a highly H(2)O-soluble fraction (would contain GA glucosyl conjugates and very polar free GAs). Quantitation after SiO(2) partition chromatography was accomplished by microdrop and immersion dwarf rice bioassays. As embryogenesis developed, the free and highly H(2)O-soluble GA-like substances, expressed on a dry weight basis, decreased (however, they increased on a per embryo basis). Chilling at 4 degrees C for 1 week greatly increased activity of free GA-like substances (per g dry weight and per embryo), it then declined over the next three weeks of chilling. Activity (per g dry weight and per embryo) in the H(2)O-soluble fraction declined throughout chilling. Activity in the GA-precursor fraction, however, increased steadily with chilling (per g dry weight and per embryo). Incubation at 26 degrees C after chilling enhanced activity in the free GA and H(2)O-soluble fractions (per g dry weight and per embryo), but activity in the GA-precursor fraction dropped dramatically. Incubation at 26 degrees C with (+/-) ABA after chilling prevented germination and maintained high activity for GA precursors and less polar free GAs and low activity in the polar free GA and H(2)O-soluble fractions.Kaurene and kaurenoic acid were characterized in the GA-precursor fraction of chilled embryos by gas-liquid chromatography-mass spectrometry (GLC-MS). The existence of GA(4) and GA(9) in ABA-treated, chilled embryos was also confirmed by GLC-MS.

Purification and separation of plant gibberellins from their precursors and glucosyl conjugates.

A procedure using two small preparative columns (in sequence) of C(18) reverse phase Bondapak B material with methanolic extracts of plant tissue (Pisum sativum L., Malus domestica Borkh., Pimpinella anisum L.) yields two fractions: (i) gibberellin (GA) precursors, and (ii) free GA/GA methyl esters (GA-Me)/GA glucosyl conjugates. The discrete separation of (iii) free GA/GA-Me from (iv) GA glucosyl conjugates is then accomplished by a combination of differential solvent solubility and SiO(2) partition chromatography. All fractions are almost pigment free, and appreciable dry weight purification was accomplished for the GA precursor and free GA/GA-Me fractions. Solvent volumes can be kept low, no buffer salts are introduced, and each fraction (i, iii, iv) can be subjected directly to preparative or analytical reverse phase C(18) high performance liquid chromatography without recourse to solvent partitioning, and often without further purification.