Human acetyl-CoA carboxylase 2. Molecular cloning, characterization, chromosomal mapping, and evidence for two isoforms.

cDNA encoding the 280-kDa acetyl-CoA carboxylase 2 (ACC2) isoform was isolated from human liver using the polymerase chain reaction. Sequencing the cDNA revealed an open reading frame of 7,449 base pairs (bp) that encode 2,483 amino acids (Mr 279,380). Using 5-kilobase pair cDNA clones as probes, we localized the gene encoding the 280-kDa human carboxylase to chromosome 12q23. When the cDNA of ACC2 was compared with that of ACC1, the nucleotide sequences and the predicted amino acid sequences had about 60 and 80% identity, respectively. Ser77 and Ser79, which were found to be critical for the phosphorylation and subsequent inactivation of rat ACC1 (Ser78 and Ser80 of human ACC1), are conserved in ACC2 and are represented as Ser219 and Ser221, respectively. On the other hand, Ser1200, which is also a phosphorylation site in rat ACC1 (Ser1201 of human ACC1), is not conserved in ACC2. The homology between the amino acid sequences of the two human carboxylases, however, is primarily found downstream of residues Ser78 and Ser81 in human ACC1 and their equivalents, that is Ser219 and Ser221 in ACC2, suggesting that the sequence of the first 218 amino acids at the N terminus of ACC2 represents a unique peptide that accounts, in part, for the variance between the two carboxylases. Using a cDNA probe (400 bp) that encodes the N-terminal amino acid residues of ACC2 in Northern blot analyses of different human and mouse tissues showed that ACC2 is predominantly expressed in liver, heart, and the skeletal muscles. Polyclonal antibodies raised against the N-terminal peptide (amino acid residues 1-220) reacted specifically and equally with human and rat ACC2 carboxylases, confirming the uniqueness of this N-terminal peptide and its conservation in animal ACC2. In addition, we present evidence for the presence of an isoform of ACC2 (Mr 270,000) in human liver that differs from the 280-kDa ACC2 by the absence of 303 nucleotides that encode 101 amino acids in the region between Arg1114 and Asp1215. The regulation and physiological significance of the two ACC2 isoforms remain to be determined.

[Behavior of Streptococcus mutans under prolonged exposure to xylitol, without reculture]. / Comportamiento de Streptococcus mutans bajo exposición prolongada a xilitol, sin recultivo.

Previous studies carried out in our laboratory showed that Streptococcus mutans--a cariogenic oral bacteria--did not metabolize an anticariogenic sugar alcohol, xylitol, even after a 10-month adaptative period with monthly transfers to a fresh medium. Due to the potential risk to adaptation observed in bacterias, it was studied the behavior of Streptococcus mutans (Strain 1161, Ingbritt) exposed to the polyol during 7 months, without monthly transfers to a new medium. After 7 months the cells were monthly transferred to a fresh medium for 7 more months. The cells were maintained and grown in a Trypticase-soytone-base medium without dextrose which contained xylitol (0.25 g/100 mL) or no sugar added. The control was represented by cells of Streptococcus mutans growing in Trypticase- soytone-base medium containing dextrose (0.25 g/100 mL). The growth pattern in the presence of xylitol was similar to that obtained in the cultures without sugar added, but it was 63-78% lower when compared with the control. The final pH in the cultures with xylitol was around 6.0; in the control it was very low (4.69 +/- 0.12). When the cells maintained in xylitol were transferred to the medium containing dextrose, the growth pattern was similar to that of the control. Any cultures fermented xylitol; the pH of the fermentation medium remained around 6.00 when the xylitol was present. No uptake of 14C-xylitol was observed and the activity of the enzyme xylitol dehydrogenase could not be detected with the experimental procedure used. The present study confirmed the Streptococcus mutans inability to metabolize xylitol, even after a prolonged adaptative period in the sugar alcohol. Moreover, it demonstrated that xylitol could be considered an inert substance to Streptococcus mutans since the cells were viable in the presence of the xylitol, or in the medium without sugar added.