Editor's note: Science Scan is a roundup of recently published biotechnology-relevant research.
Addiction to cocaine - as to nicotine and other drugs of abuse - presumably involves stubborn changes in the brain, which ease the path to relapse despite an addict's earnest efforts to abstain. But a tricky strategy called extinction training reduces the propensity for relapse, in rats and people. It takes the form of inhibitory learning that gradually cuts down cocaine-seeking behavior - in the absence of the drug itself.
In rats, the sneaky procedure calls for the same environment, and the same levers to press, as when a cocaine reward is given. But the eager rodent quickly discovers that in extinction training there is no cocaine premium. Human addicts have been treated similarly - going through the old routines of cocaine intake without the active drug.
A plausible explanation for what makes this anti-relapse process succeed is revealed in Nature, dated Jan. 2, 2003. Its title: "Extinction-induced upregulation in AMPA receptors reduces cocaine-seeking behavior." The paper's authors are molecular psychiatrists at Yale University School of Medicine in New Haven, Conn. (AMPA stands for "amino-3-hydroxy-5-methyl-4-isoxazole propionate.")
Extinction training in rats, the paper reports, caused AMPA-type glutamate receptors to increase in the nucleus accumbens shell, a region of the brain critically involved in cocaine reward. That finding boosts the notion that behavior-based treatment strategies can be used as alternatives to pharmaceutical approaches to reverse the harmful effects of cocaine on the brain. It also points to ways in which that strategy might be mimicked pharmacologically, especially to diminish the tendency to relapse under stressful situations after prolonged abstinence.
A single extinction training session, when conducted during glutamate receptor (GluR) subunit overexpression, attenuated stress-induced relapse to cocaine seeking even after GluR declined. In the co-authors' in vivo rodent experiment, rats were trained to press a lever to receive intravenous cocaine injections for four hours daily during 12 days. After self-administration of the reward, they were allowed to extinguish cocaine-seeking responses in the absence of cocaine for four hours a day in the self-administration chambers, or were confined to their home cages for the same withdrawal period.
In animals that underwent extinction training, AMPA receptor subunits GluR1 and GluR2/3 went up by 39 and 31 percent, respectively, in the nucleus accumbens shell region. These upticks were relative to both untreated controls and cocaine-trained rats that remained in their home cages during withdrawal.
"Our results," the co-authors conclude in their Nature paper, "indicate that AMPA upregulation may benefit cocaine addicts both directly - by facilitating control over the urge for cocaine - and indirectly, by attenuating stress-induced cravings during prolonged abstinence. In addition, they suggest that behavior-based approaches have the potential to reverse or ameliorate the harmful neurobiological and behavioral consequences of chronic drug use."
Cloned Pigs With Rejection Enzymes Knocked Out Step Up To Donor Xenotransplant Plate
Except for one pesky enzyme named alpha 1,3-GT (galactosyltransferase), pigs - or rather piglets - could become man's (and woman's) best friends. This finicky molecule perches on the cell surface of every mammal on earth, other than human beings, apes and Old World monkeys. Except for these exceptions, piglets would be likely candidates as organ donors for xenotransplantation into human recipients. Porcine hearts and kidneys would be of apt size and function to replace cardiac and renal organs - but for the presence of alpha 1,3 GT all over their cells. With these enzymatic passengers stowed away on board, a piglet's donor organ would burst out in hyperacute rejection (HAR).
A team led by PPL Therapeutics Inc. has picked up this gauntlet. Their report in Sciencexpress dated Dec. 19, 2002, announces: "Production of a1,3-galactosyltransferase-deficient pigs."
The co-authors point out, "A bacterial toxin can be used to select cultured cells lacking both copies of an unwanted gene." They began with porcine cells lacking one copy of the gene that codes for the HAR-threatening enzyme. This molecule produces certain cell-surface sugars that are obstacles to xenotransplantation. Accordingly, the team added a toxin from the bacterium Clostridium difficile, which killed any cells carrying the sugar. Although the co-authors used a targeting protocol to knock out the second gene copy, they did select for a single nucleotide mutation. This would be an advantage, they noted, as it was not based on antibiotics or antibiotic resistance genes, thus making it safe for xenotransplantation.
Sequencing analysis showed that knockout of the gene's second allele was due to a single point mutation in exon 9, which inactivated alpha 1,3-GT. Four healthy double-KO female piglets were produced by three consecutive rounds of cloning. The team transferred embryos to five recipient gilts, and four normal-sized fetuses were recovered. To confirm that the mutated cDNA could not make functional 1,3-GT protein, they transferred clones into cultured human HeLa cells, which do not express the offending protein.
Oops! Factor' May Cause Rapidly Dividing Cells To Go Fragile On Pathway To Cancer
What with 46 chromosomes and 6 feet of DNA to copy every time most human cells divide, it's not surprising that gaps or breaks sometimes show up in the finished product - especially when the cell is dividing rapidly, as in cancer. Scientists call these gaps or breaks "fragile sites," but the reasons for their instability remained a mystery.
Now, geneticists at the University of Michigan in Ann Arbor have discovered that a protein called ATR protects fragile sites from breaking during DNA replication. Results of their finding appear in the Dec. 13, 2002, issue of Cell, under the title: "ATR regulates fragile site stability."
The discovery offers the first evidence of a major molecular pathway that regulates genomic stability at chromosomal fragile sites. Since these breaks are very common in some tumor cells, and often occur near genes associated with tumors, defects in the ATR protein pathway are thought to be involved in the progression of cancer.