There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoesaconformationalchange,causingtemperaturedependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation.The constitutional t(11;22)(q23;q11) is the only known recurrent non-Robertsonian translocation in humans. Its recurrent nature implicates a specific genomic structure at the t(11;22) breakpoints. Analyses of numerous independent t(11;22) cases have localized the breakpoints within palindromic AT-rich repeats (PATRRs) 1 on 11q23 and 22q11 (1-4). Most 11;22 translocations show breakpoints at the center of the PATRRs, suggesting that the center of the palindrome is susceptible to double-strand breaks, leading to the translocation (5, 6). Indeed, translocation-specific PCR detects a high frequency of de novo t(11;22)s in normal sperm samples (7).The breakpoints on 22q11 are located within one of the unclonable gaps in the human genome (8, 9). Extensive screening of YAC/BAC/PAC libraries has not been successful in cloning this breakpoint region. However, experimentally derived sequences from numerous t(11;22) junction fragments demonstrate that the 22q11 breakpoints reside within a larger PATRR. The breakpoints of a variety of translocations involving 22q11 cluster within this region, suggesting that the 22q11 PATRR is highly unstable (10 -13). More recently, molecular cloning of translocation breakpoints has demonstrated similar palindromic sequences on partner chromosomes, such as 17q11, 4q35

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Hyperlactate production is related to disorders of the large intestine such as inflammatory bowel diseases. Lactate, an intermediate in hindgut fermentation, is metabolized to SCFA. Megasphaera elsdenii can convert lactate to butyrate, a physiologically important organic acid for the hindgut mucosa. This experiment was conducted to determine whether M. elsdenii normalizes hyperlactate production and stimulates butyrate production in the rat large intestine. Specific pathogen-free Sprague-Dawley male rats (n = 12) were fed a fructooligosaccharide (FOS)-supplemented (100 g/kg), semipurified diet to induce lactate production. Lactate excretion in all rats was >30 mmol/kg fresh feces on d 2 of FOS-feeding. The rats were divided into two groups on the morning of d 4. One group (n = 5) was dosed orally with M. elsdenii JCM1772T (1.3 x 10(13) cells) for 3 d. The other group was treated with a vehicle solution. Fecal lactate was significantly lower in rats administered M. elsdenii than in controls. An increase in fecal butyrate compensated for the decrease in lactate. The number of cecal epithelial cells was greater in rats administered M. elsdenii than in controls. M. elsdenii has the potential to normalize hyperlactate accumulation in the large intestine, and lactate-utilizing butyrate producers may be useful probiotics when hyperlactate fermentation in the large intestine is a problem.

Translocation is one of the most frequently occurring human chromosomal aberrations. Balanced carriers usually manifest no phenotype but experience problems with reproduction. These include infertility, recurrent abortion, and offspring with chromosomal imbalance. The constitutional t(11;22)(q23;q11) is a balanced translocation between chromosomes 11 and 22, with breakpoints at bands 11q23 and 22q11. It is the only known recurrent non-Robertsonian translocation and represents a good model for studying translocations in humans (1). The recurrent nature of this translocation prompted us to examine t(11;22) breakpoints for a specific genomic structure. The analysis of many unrelated t(11;22) cases revealed that the breakpoints occur within palindromic AT-rich repeats (PATRRs) on 11q23 and 22q11 (PATRR11 and PATRR22) (2-4). The majority of the breakpoints are localized at the center of the PATRRs, suggesting that the center of the palindrome is susceptible to double-strand breaks (DSBs), thereby inducing illegitimate chromosomal rearrangement (3,5). Recent findings of PATRRlike sequences at the breakpoints of other translocations support the possibility that palindromemediated chromosomal translocation is a general pathway for human genomic rearrangements (6-8).The PATRR11 is variable in size in normal healthy individuals ( Fig. 1 and table S1). The most common allele is a ~450-base pair (bp) PATRR11 (L-PATRR11) that forms a nearly perfect palindrome (5). Several types of short variants were identified (S-PATRR11) that appear to be derived from the longer version primarily by deletion near the symmetric center of the palindromic structure. We can classify the S-PATRR11s into four groups. The most frequent 350-bp variant, S1-PATRR11, has a 50-bp deletion at both of the palindromic arms but still remains completely symmetrical. S2-PATRR11 has an asymmetric deletion at its center, but the new center manifests a symmetric palindrome. S3-PATRR11 does not possess palindromic features by virtue of a deletion at the center of the palindrome. We identified a rare 434-bp S-PATRR11, which sustained an asymmetric central deletion followed by the insertion of an ATrich sequence of unknown origin (S4-PATRR11). We also identified another rare allele with a duplication of the proximal arm, which constitutes a 603-bp asymmetric palindrome (EL-PATRR11). On the basis of the palindrome-mediated mechanism of the translocation, it is reasonable to hypothesize that the polymorphism of the PATRR11 could affect the frequency of de novo t(11;22) translocations.

We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes.