Morphogenic activity of fibroblast growth factor-2 on primary neural precursor cells in three-dimensional culture

Mouse neural precursor cells (NPC) were dissociated from fetal heads at the 10th day of gestation. When clumps of NPC were cultured in collagen gel, they grew and reorganized neural tube-like structures in medium containing fetal calf serum at 10% and supplemented with insulin, transferrin, cholera toxin and selenite. However, dissociated NPC died when they were cultured in collagen gel at low density in the same medium. Addition of fibroblast growth factor-2 (FGF-2) to this culture stimulated growth of NPC and formation of neural tube-like structures. The requirement for FGF-2 disappeared in high seeding density culture: they grew and formed neural tube-like structures without FGF-2. The structures formed in collagen gel were immunohistochemically positive against anti-FGF-2 antibody. The results show that the three-dimensional culture system provides a useful tool to study the roles of FGF-2 in morphogenesis of the central nervous system.     

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl₂. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.     

Elution of dissolved Fe from mangrove soil by tannin solution

To reveal the role of tannins in mangroves, tannins in mangrove leaves and the Fe eluted from mangrove soil by adding tannin solutions of different salinity levels was investigated. Leaves of six mangrove and 16 non-mangrove species, and samples of a mangrove floor, Andosol and dark red soil were collected. Results were: (1) Increasing tannic acid concentration to ~50 mM, increased the Fe eluted from mangrove soil to ~20 μgg^?1. (2) When a 100 mM tannic acid solution was added, the Fe eluted from mangrove soil was 5.5 times higher than dark red soil. (3) Although elution of Fe from mangrove soil was higher than in Andosol one day after submersion in a 10 mM tannic acid solution, the difference was stable after 2 days. (4) The elution of Fe from all soils significantly decreased with increasing salinity of a 10 mM tannic acid solution. However, the amount from mangrove soil was 6.1 times higher than dark red soil even with 35 ‰ salinity. (5) The tannin content in the mangrove leaves was 99 ± 16 mgg^?1 and non-mangrove leaves was 76 ± 19 mgg^?1. (6) The Fe eluted from mangrove soil had a positive correlation with the tannin concentrations in the added leaf solution. Tannins in mangrove species promote the elution of Fe from mangrove floor soil even in saline water. Fe complexes were formed when mangrove soil was mixed with leaf tannins suggesting that Fe produced by tannins in mangrove leaves growing in land/sea interfaces likely plays a direct role in marine ecosystems.     

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.     

Effects of Temperature on Bud-Sprouting and Early Growth of Cyperus esculentus in the Dark

Cyperus esculentus tubers and early growth of their sprouts. Percent sprouting increased with increasing temperature within the range of 12 to 38 C, while no sprouting occurred at 10 C and few tubers sprouted at 42 C. The rate of sprouting also increased with temperature up to 35 C. A base temperature of 11.4 C was determined for bud-sprouting of tubers in this species. Higher temperatures led to larger sprouts and greater survival rate. In particular, increased temperature favored root growth, and hence resulted in high root: shoot ratio of the sprouts. Larger tubers produced larger sprouts as a consequence of mobilizing a greater amount of their reserves, but they tended to utilize a smaller proportion of their reserves. The efficiency of reserve utilization significantly differed among the incubation temperatures, and its relation with temperature followed a quadratic pattern. This pattern is different from that documented for the bud-sprouting of rhizomes and stolons of other perennials. Our results demonstrate that temperature is crucial to the successful establishment of C. esculentus. Received 24 June 1999/ Accepted in revised form 14 December 1999     

Risk Factors for Cisplatin-Induced Nephrotoxicity and Potential of Magnesium Supplementation for Renal Protection

Background

Nephrotoxicity remains a problem for patients who receive cisplatin chemotherapy. We retrospectively evaluated potential risk factors for cisplatin-induced nephrotoxicity as well as the potential impact of intravenous magnesium supplementation on such toxicity.

Patients and Methods

We reviewed clinical data for 401 patients who underwent chemotherapy including a high dose (≥60 mg/m²) of cisplatin in the first-line setting. Nephrotoxicity was defined as an increase in the serum creatinine concentration of at least grade 2 during the first course of cisplatin chemotherapy, as assessed on the basis of National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. The severity of nephrotoxicity was evaluated on the basis of the mean change in the serum creatinine level. Magnesium was administered intravenously to 67 patients (17%).

Results

Cisplatin-induced nephrotoxicity was observed in 127 patients (32%). Multivariable analysis revealed that an Eastern Cooperative Oncology Group performance status of 2 (risk ratio, 1.876; P = 0.004) and the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) (risk ratio, 1.357; P = 0.047) were significantly associated with an increased risk for cisplatin nephrotoxicity, whereas intravenous magnesium supplementation was associated with a significantly reduced risk for such toxicity (risk ratio, 0.175; P = 0.0004). The development of hypomagnesemia during cisplatin treatment was significantly associated with a greater increase in serum creatinine level (P = 0.0025). Magnesium supplementation therapy was also associated with a significantly reduced severity of renal toxicity (P = 0.012).

Conclusions

A relatively poor performance status and the regular use of NSAIDs were significantly associated with cisplatin-induced nephrotoxicity, although the latter association was marginal. Our findings also suggest that the ability of magnesium supplementation to protect against the renal toxicity of cisplatin warrants further investigation in a prospective trial.     

Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.     

Xylem water-conducting patterns of 34 broadleaved evergreen trees in southern Japan

A dye injection method was used to elucidate the xylem water-conducting pathways of 34 broadleaved evergreen trees growing in southern Japan: two semi-ring-porous, 26 diffuse-porous, five radial-porous and one non-vessel species. The large earlywood vessels in semi-ring-porous species have a water transport function in only the outermost annual ring, as in deciduous ring-porous species. On the other hand, the small vessels in semi-ring-porous species maintain the water transport function in many outer annual rings. For the other xylem-type species, the many vessels in many outer annual rings have a water transport function. In diffuse-porous species, we categorized the water-conducting pattern within the annual rings into two types: d1 type, where water travels through vessels in the whole region; and d2 type, where water travels mainly through the earlywood vessels. The pattern in radial-porous species is similar to that in the d1 type; the pattern in non-vessels species is similar to that in the d2 type. The vessel diameter in radial-porous species is similar to that of the earlywood vessels of semi-ring-porous species. These results suggest that the conduit diameter size is only one of many factors determining the water-conducting pathways of broadleaved evergreen species.