Na+/Cl(-)-dependent glycine transporters are crucial for the termination of neurotransmission at glycinergic synapses. Two different glycine transporter genes, GlyT1 and GlyT2, have been described. Several isoforms differing in their 5′ ends originate from the GlyT1 gene. We have determined the genomic structure of the murine GlyT1 gene to elucidate the genetic basis underlying the different isoforms. Analysis of cDNA 5′-ends revealed that the GlyT1a and 1b/1c mRNAs are transcribed from two different promoters. During murine embryonic development GlyT1 mRNAs were detectable by RNase protection assays as early as embryonic day E9 and reached maximal levels between E13 and E15. In situ hybridization revealed GlyT1 expression in the developing spinal cord mainly in the ventral part of the ventricular zone at E12. At later stages (E15) transcripts were also found in the lateral half of the basal and intermediate gray matter. In contrast, the second glycine transporter gene GlyT2 displayed a completely different expression pattern. At E11 it is expressed in the mantle zone, and at later stages throughout the ventral horns. In the adult rat brain and spinal cord, GlyT1 hybridization signals were found exclusively in glial cells. Our data indicate that GlyT1 is an early marker of neural development and encodes glia-specific transporter proteins.

ABSTRACT:Two sites from a humid tropical environment were studied with respect to soil water repellency caused by hydrocarbon contamination. Samples were analyzed for water repellency (molarity ethanol droplet method), total petroleum hydrocarbons, acute toxicity (Microtox) and field capacity. At both sites, water absorption times were logarithmically related to the molarity ethanol drop value (R > 0.95). In a sandy soil collected from an old separation battery which had been bioremediated, field capacity was strongly related to hydrocarbon concentration (R = 0.998); and at 10,000 mg/kg the calculated field capacity was only 75 % of the baseline. Water repellency was related to hydrocarbon concentration asymptotically and plant growth limiting values (severity > 3.0) were observed at low concentrations (2,400 mg/kg), even though toxicity was at, or below background levels. Bioremediated soil at this site had hydrocarbon concentrations only 1,300 ppm above background, but had extreme water repellency (severity = 4.6 -4.7). Soil water repellency was also measured in a clayey, organic rich floodable soil, in a multiple pipeline right-of-way colonized by water tolerant pasture and cattails. Water repellency was associated with total petroleum hydrocarbon concentration (R = 0.962), but was not related to field capacity or toxicity. In this low-lying site, the water repellency observed in the laboratory is probably not representative of field conditions: samples taken at the end of the ten week dry season (and only four days before the first rains) showed ample moisture (> 80 % field capacity).

Pseudomonas acidovorans M3GY is a recombinant bacterium with the novel capacity to utilize a biphenyl congener chlorinated on both rings, 3,4'-dichlorobiphenyl (3,4'-DCBP), as a sole carbon and energy source. Strain M3GY was constructed with a continuous amalgamated culture apparatus (L. Krickel and D. D. Focht, Appl. Environ. Microbiol. 53:2470-2475, 1987) with P. acidovorans CC1(19), a chloroacetate and biphenyl degrader, and Pseudomonas sp. strain CB15(1), a biphenyl and 3-chlorobenzoate degrader. Genetic and phenotypic data showed the recipient parental strain to be P. acidovorans CCI and the donor parental strain to be Pseudomonas sp. strain CB15. In growth experiments with 3,4'-DCBP as a sole source of carbon, cultures of strain M3GY increased in absorbance from 0.07 to 0.39 in 29 days while reaching a protein concentration of 58 ,ug ml-' and 67% substrate dehalogenation. 4-Chlorobenzoate was identified from culture supernatants of strain M3GY by gas chromatography-infrared spectrometry-mass spectrometry; this would be consistent with the oxidation of the m-chlorinated ring through the standard biphenyl pathway. 4-Chlorobenzoate was converted to 4-chlorocatechol, which was metabolized through the meta-fission pathway. The construction ofP. acidovorans M3GY, with the novel capability to utilize 3,4'-DCBP, thus involves the complete use ofmeta-fission pathways for sequential rupture of the biphenyl and chlorobenzoate rings.

Recombinant Pseudomonas sp. strain CB15, which grows on 3-chlorobiphenyl (3CB), was constructed from Pseudomonas sp. strain HF1, which grows on 3-chlorobenzoate, and from Acinetobacter sp. strain P6, which grows on biphenyl, by using a continuous amalgamated culture apparatus. DNA from strains CB15 and HF1 hybridized very strongly to each other, while hybridization between both parental strains, HF1 and P6, was negligible. However, DNA from the recombinant CB15 hybridized moderately to strongly with three specific fragments of parental strain P6. Strains HF1 and P6 did not grow on 3CB, but recombinant strain CB15 mineralized this compound and released inorganic chloride. When growing on 3CB, strain CB15 accumulated brown products, one of which was identified as 3-chloro-5-(2'-hydroxy-3'-chlorophenyl)-1,2-benzoquinone by mass spectrometry. Emulsification and mechanical fragmentation greatly increased the rate of 3CB mineralization by strain CB15. At least three methods of inhibition from catecholic intermediates may account for slow growth on 3CB. The meta fission of 2,3-dihydroxybiphenyl (the nonchlorinated analog of the metabolic intermediate 3-chloro-2',3'-dihydroxybiphenyl) was affected by substrate inhibition (Vmax = 359 nmolmin-' mg-', Km = 114 R,M, K.. [the inhibition constant] = 951 ,uM) and was also inhibited by 3-chlorocatechol. The ortho fission of 3-chlorocatechol, a degradation product, followed Michaelis-Menten kinetics (Vmax = 365 nmolmin-' * mg-', Km = 1 ,uM), but the addition of 2,3-dihydroxybiphenyl inhibited the reaction (Ki = 0.87 ,uM). In resting-cell assays, recombinant strain CB15 and strain P6 both metabolized 3CB faster than 3,3'-dichlorobiphenyl. However, 3,3'-dichlorobiphenyl could not be utilized as a growth substrate by strain CB15, nor did its presence have any effect on the rate of 3CB mineralization.

Developing countries face the challenge of growing their economy while reducing the negative environmental impacts of industry, thus requiring treatment technologies that are economical and effective. One recent technology developed in the tropical part of Mexico for the remediation of petroleum-contaminated soil was tested in this scale-up project at an industrial level, whereas previously it had only been tested at laboratory scale; 150 m 3 of bentonitic mud, contaminated with weathered hydrocarbons (3.4°API) at *50,000 ppm, was treated with 4 % Ca(OH) 2 , 4 % organic amendment, and a fine-root tropical grass. Hydrocarbons in soil and in leachates, as well as pH, and acute toxicity (Microtox) were monitored for 28.8 months. At the end of the study, basal respiration, root density, and earthworm toxicity were also measured. The hydrocarbon concentration in soil was reduced to 45 %, and toxicity was eliminated. Hydrocarbons in leachates were reduced to *1 mg/l, safe for human consumption. The pH adjustment depended on low soil moisture and was stabilized at 7.1. Intense revegetation resulted in good root density, within 90 % of nearby uncontaminated soil under pasture. Basal respiration was increased to levels comparable to uncontaminated tropical soils with agricultural use, pasture and gallery forest. At an industrial scale, strict moisture control was necessary for good pH stabilization. By controlling these conditions and applying this novel treatment process, it was possible to transform a heavily contaminated geological material into a non-toxic, fertile, soil-like substrate capable of maintaining a complete vegetative cover and microbial activity comparable to similar soils in a tropical environment.