Saami and Berbers--an unexpected mitochondrial DNA link

The sequencing of entire human mitochondrial DNAs belonging to haplogroup U reveals that this clade arose shortly after the "out of Africa" exit and rapidly radiated into numerous regionally distinct subclades. Intriguingly, the Saami of Scandinavia and the Berbers of North Africa were found to share an extremely young branch, aged merely approximately 9,000 years. This unexpected finding not only confirms that the Franco-Cantabrian refuge area of southwestern Europe was the source of late-glacial expansions of hunter-gatherers that repopulated northern Europe after the Last Glacial Maximum but also reveals a direct maternal link between those European hunter-gatherer populations and the Berbers.     

MRE11/RAD50/NBS1: complex activities

The MRE11/RAD50/NBS1 complex (Mre11 complex) is a central player in most aspects of the cellular response to DNA double-strand breaks, including homologous recombination, non-homologous end joining, telomere maintenance and DNA damage checkpoint activation. Several of these findings are explained by the unusual enzymatic activities and macromolecular structure of the Mre11 complex. The Mre11 complex possesses an ATP-stimulated nuclease to process heterogeneous DNA ends and long coiled-coil tails to link DNA ends and/or sister chromatids. However, the mechanistic role of the Mre11 complex in checkpoint activation has been unclear until recently. New data suggest that the Mre11 complex can directly activate the ATM checkpoint kinase at DNA breaks. These findings, together with newly determined functional interactions, identify the Mre11 complex as an architectural and mechanistic keystone of cellular response events emerging from DNA breaks.     

Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain

Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.     

Up-regulation of CD1d expression restores the immunoregulatory function of NKT cells and prevents autoimmune diabetes in nonobese diabetic mice

The immunoregulatory function of NKT cells is crucial for prevention of autoimmunity. The prototypical NKT cell Ag alpha-galactosylceramide is not present in mammalian cells, and little is known about the mechanism responsible for NKT cell recruitment and activation. Up-regulation of CD1d, the NKT cell restriction molecule, expressed on mononuclear cells infiltrating the target organ, could represent the physiological trigger for NKT cells to self-contain T cell immunity and to prevent autoimmune disease. Recognition of CD1d, either by itself or bound to self-ligands (selfCD1d), could drive NKT cells toward an immunoregulatory phenotype. Hence, ineffective NKT cell-mediated immunoregulation in autoimmune-prone individuals including nonobese diabetic (NOD) mice could be related to defective signals that regulate CD1d expression at time and site of autoimmunity. To test this hypothesis, we transgenically overexpressed CD1d molecules under the control of the insulin promoter within the pancreatic islets of NOD mice (insCD1d). Recognition of overexpressed CD1d molecules rescued NKT cell immunoregulatory function and prevented autoimmune diabetes in insCD1d transgenic NOD mice. Protection from diabetes was associated with a biased IL-4-secreting cytokine phenotype of NKT cells and alteration of the cytokine microenvironment in the pancreatic lymph nodes of transgenic mice. The net effect was a reduced development of the autoimmune T cell repertoire. Our findings suggest that up-regulation of CD1d expression during inflammation is critical to maintain T cell homeostasis and to prevent autoimmunity.     

Terminal deoxynucleotidyltransferase deficiency decreases autoimmune disease in diabetes-prone nonobese diabetic mice and lupus-prone MRL-Fas(lpr) mice

The wide diversity of the T and B Ag receptor repertoires becomes even more extensive postneonatally due to the activity of TdT, which adds nontemplated N nucleotides to Ig and TCR coding ends during V(D)J recombination. In addition, complementarity-determining region 3 sequences formed in the absence of TdT are more uniform due to the use of short sequence homologies between the V, D, and J genes. Thus, the action of TdT produces an adult repertoire that is both different from, and much larger than, the repertoire of the neonate. We have generated TdT-deficient nonobese diabetic (NOD) and MRL-Fas(lpr) mice, and observed a decrease in the incidence of autoimmune disease, including absence of diabetes and decreased pancreatic infiltration in NOD TdT(-/-) mice, and reduced glomerulonephritis and increased life span in MRL-Fas(lpr) TdT(-/-) mice. Using tetramer staining, TdT(-/-) and TdT(+/+) NOD mice showed similar frequencies of the diabetogenic BDC 2.5 CD4(+) T cells. We found no increase in CD4(+)CD25(+) regulatory T cells in NOD TdT(-/-) mice. Thus, TdT deficiency ameliorates the severity of disease in both lupus and diabetes, two very disparate autoimmune diseases that affect different organs, with damage conducted by different effector cell types. The neonatal repertoire appears to be deficient in autoreactive T and/or B cells with high enough affinities to induce end-stage disease. We suggest that the paucity of autoreactive specificities created in the N region-lacking repertoire, and the resultant protection afforded to the newborn, may be the reason that TdT expression is delayed in ontogeny.     

Expression and characterization of a novel CD6 ligand in cells derived from joint and epithelial tissues

CD6 is a T cell surface glycoprotein that plays an important role in interactions of thymocytes with thymic epithelial cells and in mature T cell interactions with selected nonprofessional tissue APCs. We describe a novel CD6 ligand (CD6L) 3A11 Ag that is distinct from the known CD6L (CD166). The 3A11 protein is expressed on cells derived from human thymus, skin, synovium, and cartilage, and its expression is enhanced by IFN-gamma. mAbs directed against the 3A11 Ag and CD166 exhibit distinct patterns of binding to a panel of cell lines. Confocal microscopy shows that both CD166 and the 3A11 Ag are expressed at the cell surface, and that these proteins colocalize. The 3A11 Ag has a molecular mass of 130 kDa and is immunoprecipitated using either mAb 3A11 or soluble CD6-Ig fusion protein. mAbs directed against individual CD6L were less potent than was soluble CD6-Ig fusion protein in reducing adhesion of T cells to adherent 3A11-positive epithelial cells in vitro, suggesting that these Abs recognize epitopes on the 3A11 Ag and CD166 that are distinct from CD6 binding sites. Finally, transfection of epithelial cells with CD166-specific small interfering RNAs significantly decreased CD166 expression without alteration in 3A11 Ag levels, and thus confirmed that these two CD6L are distinct. Taken together, our data identifies a novel 130-kDa CD6L that may mediate interactions of synovial and epithelial cells with T lymphocytes.     

Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-beta (TGF-beta) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-beta signaling, to identify distinct roles for this pathway in adult and embryonic beta cells. Smad7 expression in Pdx1+ embryonic pancreas cells resulted in striking embryonic beta cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1+ cells reduced detectable beta cell expression of MafA, menin, and other factors that regulate beta cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-beta signaling. Thus, our studies reveal that TGF-beta signaling is crucial for establishing and maintaining defining features of mature pancreatic beta cells.     

Thermal biology of the southernmost lizards in the world: Liolaemus sarmientoi and Liolaemus magellanicus from Patagonia,Argentina

We determined the efficiency of thermoregulation by the southernmost liolaemids Liolaemus sarmientoi and L. magellanicus from Patagonia, Argentina (51°S), by measuring body (T_b), microenvironmental, and operative temperatures in the field, and preferred body temperatures in the laboratory (T_pref). L. sarmientoi was found to be a poor thermoregulator, whereas L. magellanicus was deemed to be a constrained thermoconformer. Among all known species of Liolaemus, L. sarmientoi and L. magellanicus had the lowest T_b's when tested in the field; however, their T_pref's were similar to other liolaemids. These data suggest that these southernmost liolaemid species have not evolved appropriate thermoregulatory behaviors or made adequate physiological adaptations to face the extreme thermal challenges of their environment.     

Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.     

Genome Sequence of Streptococcus gallolyticus: Insights into Its Adaptation to the Bovine Rumen and Its Ability To Cause Endocarditis

Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is an increasing cause of endocarditis among streptococci and frequently associated with colon cancer. S. gallolyticus is part of the rumen flora but also a cause of disease in ruminants as well as in birds. Here we report the complete nucleotide sequence of strain UCN34, responsible for endocarditis in a patient also suffering from colon cancer. Analysis of the 2,239 proteins encoded by its 2,350-kb-long genome revealed unique features among streptococci, probably related to its adaptation to the rumen environment and its capacity to cause endocarditis. S. gallolyticus has the capacity to use a broad range of carbohydrates of plant origin, in particular to degrade polysaccharides derived from the plant cell wall. Its genome encodes a large repertoire of transporters and catalytic activities, like tannase, phenolic compounds decarboxylase, and bile salt hydrolase, that should contribute to the detoxification of the gut environment. Furthermore, S. gallolyticus synthesizes all 20 amino acids and more vitamins than any other sequenced Streptococcus species. Many of the genes encoding these specific functions were likely acquired by lateral gene transfer from other bacterial species present in the rumen. The surface properties of strain UCN34 may also contribute to its virulence. A polysaccharide capsule might be implicated in resistance to innate immunity defenses, and glucan mucopolysaccharides, three types of pili, and collagen binding proteins may play a role in adhesion to tissues in the course of endocarditis.Several studies have reported that the proportion of infective endocarditis due to Streptococcus gallolyticus has increased during the last decades, concomitantly with a decrease of cases due to oral streptococci (35). S. gallolyticus is now becoming the first cause of infectious endocarditis among streptococci in Europe (16). Furthermore, S. gallolyticus endocarditis is associated with rural residency, suggesting transmission from animals (29). However, the reasons for the emergence of this pathogen remain poorly understood. S. gallolyticus belongs to the Streptococcus bovis group known for more than 60 years to cause endocarditis (45). Recently, the former species S. bovis has been divided into four major species (50, 53). S. gallolyticus corresponds to S. bovis biotype I (mannitol fermentation positive), the closely related species S. pasteurianus to biotype II/2 (mannitol negative and β-glucuronidase positive), and the more distantly related species S. infantarius to biotype II/1 (mannitol negative and β-glucuronidase negative). S. macedonicus, the fourth species, commonly found in cheese, is nonpathogenic and also considered a S. gallolyticus subspecies (53, 62). A majority of endocarditis cases was due, among the formerly S. bovis group, to S. gallolyticus strains (4).Multiple studies have shown that endocarditis due to S. gallolyticus as well as positive blood culture for this species is often associated with gastrointestinal malignancy (4, 6). This association has led to a strong indication for gastrointestinal investigation and endoscopic follow-up in the case of S. gallolyticus infections (66). The association of S. gallolyticus infection with colon cancer is a major but still unsolved issue. It may be just incidental, as the alteration of the digestive mucosa may favor the translocation of the bacteria into the bloodstream. Alternatively, the tumor may contribute to the proliferation of S. gallolyticus in close proximity to the gut epithelium, increasing its probability of translocating through the gut barrier. It has also been suggested that the bacterium itself contributes to carcinogenesis (60, 69). In addition to human disease, S. gallolyticus may also cause diseases in animals, like septicemia in pigeons (19), outbreaks in broiler flocks (11), or bovine mastitis (28).Independent from its association to disease, S. gallolyticus has been isolated as a tannin-resistant bacterium from the feces of different mammalian herbivores, including the koala (48) or the Japanese large wood mouse (52), and it is also a normal inhabitant of the rumen (39). Its resistance to tannins is linked to its tannase activity, a characteristic which also led this bacterium to be named “gallolyticus” as it is able to decarboxylate gallate, an organic acid derived from tannin degradation. S. gallolyticus is also known to express other degradative functions unique among streptococci, like a bile salt hydrolase or an amylase. These properties allow its multiplication outside the animal host, as S. gallolyticus was isolated from a digester fed with shea cake (derived from the nuts of the African tree Vitellaria paradoxa) rich in tannins and aromatic compounds (12). S. gallolyticus is a commensal of the human intestinal tract but remains a rarely detected (2.5 to 15%) low-abundance species (10, 40). In herbivores, overgrowth of S. bovis may become deleterious. For example, ingestion of large amounts of rapidly fermented cereal grains leads to a destabilization of the rumen flora and to the proliferation of acid-tolerant bacteria, including S. gallolyticus. This is accompanied by the overproduction of mucopolysaccharides that stabilize the foam, resulting in feedlot bloat, a significant cause of economical loss (14).Virulence and colonization factors of S. gallolyticus in humans are largely unknown. Studies of the bird host have shown that this Streptococcus species expresses a capsular polysaccharide, and five different serotypes have been described (19). In addition, electron microscopy studies have revealed the presence of fimbria-like structures on the surface of S. gallolyticus. It was hypothesized that capsules and/or fimbriae are involved in virulence (63). S. gallolyticus isolates responsible for endocarditis exhibited heterogeneous patterns of adherence to extracellular matrix (ECM) proteins, which suggests that they produce different surface components (55). Recently, a collagen binding adhesin together with 10 putative ECM binding proteins were identified in the draft genome sequence of a human isolate of S. gallolyticus (54).Here we describe the sequence and analysis of the genome of S. gallolyticus strain UCN34 isolated from a human case of endocarditis associated with colon cancer. Analysis of the predicted proteins revealed unique metabolic and cell surface features among streptococci, which contribute to its adaptation to the rumen and to its ability to cause endocarditis. We showed by comparative genomics that many of the corresponding genes were probably acquired by lateral gene transfer (LGT) from other Firmicutes of the gut microbiota.