Influx mechanism of 2',3'-dideoxyinosine and uridine at the blood-placenta barrier.

The blood-placenta barrier (BPB) serves to protect the fetus from exposure to toxins, and to transport various nutrients, including nucleosides, and hormones from mother to fetus. It is known that nucleoside transporters contribute to the transfer of nucleosides and nucleoside analogues. 2',3'-Dideoxyinosine (ddI) has a nucleoside structure, and crosses the BPB. Although ddI is a substrate of several transporters, including equilibrative nucleoside transporters (ENT1 and ENT2), the transport mechanism of ddI in the placenta has not yet been characterized. Therefore, the purpose of this study was to clarify the influx mechanisms of ddI from the maternal to the fetal side, and to examine the interaction between ddI and uridine transport at the BPB. We studied ddI and uridine uptakes using a conditionally immortalized rat syncytiotrophoblast cell line, TR-TBT 18d-1, as a BPB model. The ddI uptake was temperature-dependent, Na(+)-independent and saturable. Kinetic analysis yielded K(m) values for ddI and uridine of 6.51 mM and 23.4 microM, respectively. Uridine uptake was inhibited by ENT1 and ENT2 substrates, and ddI uptake was also inhibited by substrates or inhibitors at concentrations that inhibit ENT2. Uridine uptake in Xenopus laevis oocytes expressing rat ENT2 was inhibited by 5mM ddI, in agreement with the results for TR-TBT 18d-1. Our results indicate that ddI and uridine are both taken up in part via ENT2 in TR-TBT 18d-1 cells, and therefore that ENT2 may contribute to their uptake at the BPB.

Characterization of the mechanism of zidovudine uptake by rat conditionally immortalized syncytiotrophoblast cell line TR-TBT.

PURPOSE: To characterize the uptake mechanism of zidovudine (AZT), a nucleoside reverse transcriptase inhibitor, in syncytiotrophoblast cells using the TR-TBT 18d-1 cell line previously established by our group. MATERIALS AND METHODS: The effects of several transporter inhibitors on the initial and steady-state apical uptake of AZT by TR-TBT 18d-1 were characterized, in order to identify the transporter(s) involved. RESULTS: Initial uptake of AZT was sodium-independent and saturable; the K(m) value was about 16 microM. Nitrobenzylthioinosine (NBMPR), probenecid and cimetidine each had little effect on the saturable AZT uptake, indicating that well characterized transporters, such as organic anion transporters (OATs and OATPs), organic cation transporters (OCTs) and equilibrative nucleoside transporters (ENTs), are not involved. However, thymidine and 2'-deoxyuridine strongly inhibited AZT uptake. These results suggest that an unidentified nucleoside uptake transporter is responsible for the uptake of AZT. Cyclosporin A, Ko143 and probenecid had little effect on AZT accumulation by TR-TBT 18d-1 cells, suggesting that transporter-mediated efflux of AZT is not substantial. CONCLUSION: Our results indicate that saturable AZT uptake into TR-TBT 18d-1 is mediated by a so-far-unidentified transporter.

Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120.

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.

Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

The complete nucleotide sequence of the genome of a symbiotic bacterium Mesorhizobium loti strain MAFF303099 was determined. The genome of M. loti consisted of a single chromosome (7,036,071 bp) and two plasmids, designated as pMLa (351,911 bp) and pMLb (208, 315 bp). The chromosome comprises 6752 potential protein-coding genes, two sets of rRNA genes and 50 tRNA genes representing 47 tRNA species. Fifty-four percent of the potential protein genes showed sequence similarity to genes of known function, 21% to hypothetical genes, and the remaining 25% had no apparent similarity to reported genes. A 611-kb DNA segment, a highly probable candidate of a symbiotic island, was identified, and 30 genes for nitrogen fixation and 24 genes for nodulation were assigned in this region. Codon usage analysis suggested that the symbiotic island as well as the plasmids originated and were transmitted from other genetic systems. The genomes of two plasmids, pMLa and pMLb, contained 320 and 209 potential protein-coding genes, respectively, for a variety of biological functions. These include genes for the ABC-transporter system, phosphate assimilation, two-component system, DNA replication and conjugation, but only one gene for nodulation was identified.

Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions.

The sequence determination of the entire genome of the Synechocystis sp. strain PCC6803 was completed. The total length of the genome finally confirmed was 3,573,470 bp, including the previously reported sequence of 1,003,450 bp from map position 64% to 92% of the genome. The entire sequence was assembled from the sequences of the physical map-based contigs of cosmid clones and of lambda clones and long PCR products which were used for gap-filling. The accuracy of the sequence was guaranteed by analysis of both strands of DNA through the entire genome. The authenticity of the assembled sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA using the assembled sequence data. To predict the potential protein-coding regions, analysis of open reading frames (ORFs), analysis by the GeneMark program and similarity search to databases were performed. As a result, a total of 3,168 potential protein genes were assigned on the genome, in which 145 (4.6%) were identical to reported genes and 1,257 (39.6%) and 340 (10.8%) showed similarity to reported and hypothetical genes, respectively. The remaining 1,426 (45.0%) had no apparent similarity to any genes in databases. Among the potential protein genes assigned, 128 were related to the genes participating in photosynthetic reactions. The sum of the sequences coding for potential protein genes occupies 87% of the genome length. By adding rRNA and tRNA genes, therefore, the genome has a very compact arrangement of protein- and RNA-coding regions. A notable feature on the gene organization of the genome was that 99 ORFs, which showed similarity to transposase genes and could be classified into 6 groups, were found spread all over the genome, and at least 26 of them appeared to remain intact. The result implies that rearrangement of the genome occurred frequently during and after establishment of this species.

Biological activity of des-asp1-angiotensin II (angiotensin III) in man.

Effects of des-asp1-angiotensin II (angiotensin III) on blood pressure and aldosterone secretion were examined in man. Angiotensin III was equipotent with val5-angiotensin II amide in the stimulation of aldosterone production, but had only 20% of the pressor activity of the later. These results are consistent with those previously reported by other investigators in animals.