Ultrasound-induced physiological effects and secondary metabolite (saponin) production in Panax ginseng cell cultures.

This work was aimed at the effects of ultrasound (US) on the growth and secondary metabolite biosynthesis of cultured plant cells. Suspension cultures of Panax ginseng cells were exposed to US at power density below 82 mW/cm3 for short periods of time (1-4 min) in a US bath (38.5-kHz fixed frequency and 810 W maximum peak power). Under most exposure conditions, US stimulated the biosynthesis of secondary metabolites, the ginsenoside saponins of ginseng cells, increasing the total saponin content of the cell by up to 75%. The growth and viability of ginseng cells were usually depressed immediately after the exposure to US, but recovered gradually to levels similar to those of a normal culture in a few days, with virtually no net loss of biomass yield at the end of the culture period. At some lower US doses, sonicated cultures could even reach slightly higher biomass yields than that of normal cultures. The effects of US on cell growth and secondary metabolite yield showed a significant correlation with the total US energy emitted (i.e., the product of US power and exposure time). Mechanical stress and microstreaming induced by acoustic cavitation were considered as the most possible causes of the various physiological effects of US on ginseng cells. In particular, the stimulation of secondary metabolite production by US may be a result of US-induced plant cell defense response.

PCR cloning of type II polyhydroxyalkanoate biosynthesis genes from two Pseudomonas strains.

Two polyhydroxyalkanoate synthase genes, phaC1 from Pseudomonas pseudoalcaligenes HBQ06 and phaC2 from Pseudomonas nitroreducens 0802, were cloned using a PCR cloning strategy based on the type II pha loci property of Pseudomonas strains. The complete open reading frames (ORFs) of phaC1 (P. nitroreducens HBQ06) and phaC2 (P. nitroreducens 0802) were identified from the PCR products. Using the sequence information, the complete PHA synthase genes were PCR cloned directly from the genomic DNA and expressed in Escherichia coli as confirmed by Fourier transform-infrared spectroscopy and gas chromatography. The differences between PhaC1 and PhaC2 were analyzed and the two proteins were suggested to contain different functions and evolution history.

Exercise decreases cytosolic aconitase activity in the liver, spleen, and bone marrow in rats.

Effects of strenuous exercise on cytosolic aconitase activity (CAA) were investigated in this study. Female Sprague-Dawley rats were randomly assigned to four groups: S1 (Sedentary), S2 (Sedentary + L-NAME [N-nitro-l-arginine methyl ester]), E1 (Exercise), and E2 (Exercise + L-NAME). Rats in the E1 and E2 groups swam for 2 h/day for 3 months. L-NAME (an inhibitor of NOS) in drinking water (1 mg/ml) was administered to rats in the S2 and E2 groups for the same period. At the end of the third month, the CAA in the liver, spleen, and bone marrow cells was measured. In the exercise group (E1), CAA in the liver, spleen, and bone marrow cells was 19.99 +/- 1.49, 1.61 +/- 0.13, and 0.59 +/- 0.09 mU/mg protein, respectively. These values were significantly lower than the corresponding sedentary values in the S1 group (33.96 +/- 1.38, 3.96 +/- 0.19, and 3.20 +/- 0.18 mU/mg protein) (P < 0.01, 0.001, and 0.001, respectively). The treatment of L-NAME led to a significant increase in tissue CAA in the sedentary rats (S2). Also, the significantly higher CAA in the liver, spleen, and bone marrow cells was found in the exercised rats treated with L-NAME (E2) (29.50 +/- 1.27, 2.89 +/- 0.25, and 1.34 +/- 0.20 mU/mg) than without L-NAME (E1) (P < 0.01, 0.01, 0.05, respectively). However, the values in the E2 group were still significantly lower than those in the S1 group (P < 0.05, 0.01, and 0.001, respectively). This indicates that L-NAME treatment can partly recover the decreased CA in tissues in the exercised rats. These results provide evidence for the existence of the increased activity of IRP1 (iron regulatory protein 1) that is probably induced by the increased nitric oxide production in the strenuously exercised rats.

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media.

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45 degrees C and in 250 g/l molasses although the optimal growth temperature was 35-37 degrees C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25-35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.

Transferrin-bound and transferrin free iron uptake by cultured rat astrocytes.

Previously we had demonstrated the presence of transferrin receptor (TfR) on the plasma membrane of cultured rat cortical astrocytes. In this study, we investigated the roles of TfR in transferrin-bound iron (Tf-Fe) as well as transferrin-free iron (Fe II) uptake by the cells. The cultured rat astrocytes were incubated with 1 microM of double-labelled transferrin (125I-Tf-59Fe) in serum- free DMEM F12 medium or 59Fe II in isotonic sucrose solution at 37 degrees C or 4 degrees C for varying times. The cellular Tf-Fe, Tf and Fe II uptake was analyzed by measuring the intracellular radioactivity with gamma counter. The result showed that Tf-Fe uptake kept increasing in a linear manner at least in the first 30-min. In contrast to Tf-Fe uptake, the internalization of Tf into the cells was rapid initially but then slowed to a plateau level after 10 min. of incubation. The addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake. Pre-treated cells with trypsin inhibited significantly the cellular uptake of Tf-Fe as well as Tf. These findings suggested that Tf-Fe transport across the membrane of astrocytes is mediated by Tf-TfR endocytosis. The results of transferrin-free iron uptake indicated that the cultured rat cortical astrocytes had the capacity to acquire Fe II. The highest uptake of Fe II occurred at pH 6.5. The Fe II uptake was time and temperature dependent, iron concentration saturable, inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+ and not significantly affected by phenylarsine oxide treatment. These characteristics of Fe II uptake by the cultured astrocytes suggested that Fe II uptake is not mediated by TfR and implied that a carrier-mediated iron transport system might be present on the membrane of the cultured cells.

Conversion of industrial food wastes by Alcaligenes latus into polyhydroxyalkanoates.

Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. As the first step in our pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesise environmental-friendly bioplastics, we investigated the usage of soya wastes from a soya milk dairy, and malt wastes from a beer brewery plant as the carbon sources for the production of polyhydroxyalkanoates (PHA) by selected strain of microorganism. Bench experiments showed that Alcaligenes latus DSM 1124 used the nutrients from malt and soya wastes to biosynthesise PHAs. The final dried cell mass and specific polymer production of A. latus DSM 1124 were 32g/L and 70% polymer/cells (g/g), 18.42 g/L and 32.57% polymer/cell (g/g), and 28 g/L and 36% polymer/cells (g/g), from malt waste, soya waste, and from sucrose, respectively. These results suggest that many types of food wastes might be used as the carbon source for the production of PHA.

Assessment of various carbon sources and nutrient feeding strategies for Panax ginseng cell culture.

Ginseng (root of Panax ginseng C. A. Meyer) cells were cultivated on medium supplemented with various carbohydrates including sucrose, glucose, and fructose, at initial concentrations ranging from 10 to 110 g/L. Sucrose was shown to be the superior carbon source to the monosaccharides for ginseng cell growth and the optimal concentration was between 30 and 50 g/L. An increase in the initial concentration within this range increased the maximum cell density and growth index significantly, whereas much higher concentrations inhibited cell growth. Feeding of sucrose and some other medium components during the growth (fed-batch mode) was more effective in enhancing the cell growth and biomass productivity, increasing the growth index by more than 60-70% and biomass productivity by more than 50%.

Identification of the major sources of error in estrogen receptor measurements for individual laboratories using both tissue and cytosol samples.

The major sources of error between laboratories performing estrogen receptor measurements in tissue samples were identified for 17 participating laboratories in a trial conducted by the Australasian Quality Assurance programme. Both tissue and cytosol samples were provided, and the In-House assays were compared with the ER-EIA kit (Abbott Laboratories, U.S.A.) as a reference assay. For both the In-House and Abbott assays, tissue samples resulted in a between laboratory CV of about 55% and a within laboratory CV of about 30%. In contrast to tissue samples, the between laboratory CV for cytosol samples was reduced to 41% for the In-House assays and to 33% for the Abbott assay, whereas the within laboratory CV was reduced to 10% for both types of assay. The different methods of tissue homogenization by themselves were not found to be sources of error, and protein extraction efficiency from tissue was strongly correlated with protein measurement (P less than 0.0005). The major sources of error due to protein measurement, cytosol preparation, In-House and Abbott assays were evaluated for individual laboratories. The results indicated absence of any major sources of error for four laboratories, while one, two and three or more sources were indicated for seven, three and three laboratories respectively. The conclusion that about half the participants need to improve their ER assays was confirmed by three independent reviews. Furthermore, the trial demonstrated that tissue samples are essential as quality assurance material for a realistic assessment of ER assays in biopsy specimens.

Effect of maternal methadone administration on the development of multiple forms of monoamine oxidase in rat brain and liver.

The development of total monoamine oxidase (MAO) and MAO-A and MAO-B in the forebrain, brainstem, cerebellum and liver of methadone-treated and normal rats were monitored with tryptamine, serotonin and benzylamine, respectively. Daily administration of methadone (10 mg/kg, s.c.) to pregnant and nursing rats substantially retarded the development of total and the A-form of MAO in the brain regions of pups but had no effect on that of MAO-B. The effect of methadone on the development of total MAO and MAO-A in the liver was only transient and less significant. This finding indicates that the perinatal opiate syndrome associated with maternal exposure to methadone is caused by the inhibition in MAO-A development in monoaminergic neurons in the brain.

Ontogenesis of multiple forms of monoamine oxidase in rat brain regions and liver.

The postnatal development of total, type-A and type-B monoamine oxidase (MAO) in the brain stem, forebrain and cerebellum, determined with preferred substrates or selective inhibitors, were found to follow different patterns. In the brain regions, MAO-A activity reached adult levels in the brain stem first, followed by the forebrain and cerebellum, while MAO-B reached adult levels in these regions at about the same time and later in postnatal life. On the other hand, both MAO-A and B activities were almost fully developed in the newborn liver. Moreover, total and type-A, but not type-B, showed a caudal-to-rostral sequence of biochemical maturation in the brain. The spatiotemporal pattern of differentiation of type-A and type-B activities in the brain tends to support the classification of brain MAO into two distinct isoenzymic forms.

Ontogenesis of opiate binding sites and radioimmunoassayable beta-endorphin and enkephalin in regions of rat brain.

The postnatal changes in the levels of radioimmunoassayable enkephalin and beta-endorphin, as well as the densities of [3H]methionine-enkephalin and [3H]naloxone binding sites in rat cerebellum, brainstem and whole forebrain were determined. The opiate peptides and the opiate binding sites reached their highest levels at the first week postpartum in the cerebellum, at the second week in the brainstem and at the third week in the whole forebrain. This finding is in line with the developmental profiles of other well-established neuronal pathways which also showed a caudal-to-rostral sequence of development. Moreover, there was a close relationship between the elevation and decline in the amounts of opiate binding sites and in the levels of opiate peptides in each brain region. These observations are consistent with other evidence which suggests that enkephalin and beta-endorphin are functioning as neurotransmitters or neuromodulators in the central nervous system.

Development of methionine-enkephalin and naloxone binding sites in regions of rat brain.

The development of opiate receptors in whole forebrain, brain stem and cerebellum of developing rats was measured by specific [3H]methionine-enkephalin ([3H]Met-Enk) and [3H]naloxone binding. The level of opiate receptor as determined by the binding of these two opiates varied with brain region as well as with age; however, the amount of [3H]naloxone bound in the same brain region obtained from animals of the same age was greater than that of [3H]Met-Enk. Kinetic analyses of these two opiate binding sites in newborns of the same age revealed that they differed in their apparent affinities and receptor numbers. Moreover, both morphine and naloxone were equally active in competing for [3H]Met-Enk binding in all 3 brain regions during the first month after birth, whereas methionine-enkephalin was only about 30-50% as active as morphine n competing for [3H]naloxone binding in that period. These results suggest that in the rat brain there are two types of opiate receptors and that the heterogeneity of opiate receptors is already apparent during early postnatal life.

Estrogen sulfurylation as an alternative indicator of hormone dependence in human breast cancer.

Estrogen receptor (ER) levels, formation of 3'-phosphoadenosine-5'phosphosulfate (PAPS), and sulfate transfer to estradiol-178, have been determined in 62 human primary carcinoma cytosol preparations. Whilst no differences between ER positive and ER negative tumors were found in the synthesis of [35S]PAPS, formation of estradiol-3-[35S]sulfate catalysed by estrogen sulfotransferase was significantly higher in ER positive tumors [84 +/- 10 (mean +/- S.E.M.) versus 32 +/- 9 pmole estradiol-3-sulfate per mg protein per 2 hr for ER positive and ER negative tumors, respectively, p less than 0.02]. By choosing a discriminating value for estrogen sulfurylation of 40 pmole per mg protein per 2 hr, then 16/19 (84%) of the ER negative tumors were below this point. Since previous data had shown that 20/23 (87%) progesterone receptor negative tumors were also below this value (Pewnim, Adams and Ho, CANCER RES. 40, 1360 (1980), an alternative simple method, having a high potential for the evaluation of hormone responsiveness in mammary cancer, is suggested.

A relationship between estrogen sulfurylation and estrogen and progesterone receptor status in human mammary carcinoma.

It was previously demonstrated that a correlation existed between estrogen receptor (ER) status and levels of estrogen sulfotransferase in human mammary cancer, high levels being associated with ER-positive and low levels with ER-negative tumors. We have now examined these same parameters, along with progesterone receptor (PGR) status, in 44 primary human mammary tumors. Tumors which were both ER-positive and PGR-positive (n = 21), showed significantly higher levels of estrogen sulfotransferase compared to ER-positive PGR-negative (n = 12) and ER-negative PGR-negative (n = 11) tumors. These values (pmol estradiol sulfate per mg protein per 2 hr) were 72 +/- 12 (S.E.), 25 +/- 5, and 11 +/- 4 (p less than 0.01 and less than 0.005, respectively). There was no significant difference between ER-positive PGR-negative and ER-negative PGR-negative tumors. The possible involvement of PGR in the regulation of estrogen sulfotransferase is discussed.

Use of values for calcium and protein in serum, and of a derived index obtained from a probability population sample.

We measured serum protein and calcium concentrations in 2340 individuals between 10 and 96 years of age from 900 families chosen by probability methods to give a representative population. These values were used to calculate an index, based on a regression analysis of serum protein on calcium, which was then treated as a new variable. Age-sex specific reference values and frequency distributions are presented for this index as well as for protein and calcium calculated by both parametric and nonparametric methods.