The Expanded mtDNA Phylogeny of the Franco-Cantabrian Region Upholds the Pre-Neolithic Genetic Substrate of Basques

The European genetic landscape has been shaped by several human migrations occurred since Paleolithic times. The accumulation of archaeological records and the concordance of different lines of genetic evidence during the last two decades have triggered an interesting debate concerning the role of ancient settlers from the Franco-Cantabrian region in the postglacial resettlement of Europe. Among the Franco-Cantabrian populations, Basques are regarded as one of the oldest and more intriguing human groups of Europe. Recent data on complete mitochondrial DNA genomes focused on macrohaplogroup R0 revealed that Basques harbor some autochthonous lineages, suggesting a genetic continuity since pre-Neolithic times. However, excluding haplogroup H, the most representative lineage of macrohaplogroup R0, the majority of maternal lineages of this area remains virtually unexplored, so that further refinement of the mtDNA phylogeny based on analyses at the highest level of resolution is crucial for a better understanding of the European prehistory. We thus explored the maternal ancestry of 548 autochthonous individuals from various Franco-Cantabrian populations and sequenced 76 mitogenomes of the most representative lineages. Interestingly, we identified three mtDNA haplogroups, U5b1f, J1c5c1 and V22, that proved to be representative of Franco-Cantabria, notably of the Basque population. The seclusion and diversity of these female genetic lineages support a local origin in the Franco-Cantabrian area during the Mesolithic of southwestern Europe, ∼10,000 years before present (YBP), with signals of expansions at ∼3,500 YBP. These findings provide robust evidence of a partial genetic continuity between contemporary autochthonous populations from the Franco-Cantabrian region, specifically the Basques, and Paleolithic/Mesolithic hunter-gatherer groups. Furthermore, our results raise the current proportion (≈15%) of the Franco-Cantabrian maternal gene pool with a putative pre-Neolithic origin to ≈35%, further supporting the notion of a predominant Paleolithic genetic substrate in extant European populations.     

Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes.     

A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

Combined effects of chlorpromazine and high fat diet on lipid levels and enzyme activities in bile, liver and plasma of Wistar rats

The effects of high fat diet and injection of chlorpromazine on bile lipid secretion were studied in the rats fed a control diet (C), a saturated fat, high cholesterol diet (S) and a polyunsaturated fat, high cholesterol diet (PU). As compared to controls, injection of chlorpromazine in the S and PU diet groups caused no appreciable change in the level of bile salts and bile phospholipids. Chlorpromazine did however enhance bile cholesterol, especially in the PU group, and lower secretion of lysosomal enzyme (beta-glucuronidase) into bile. Impairment of lysosomal enzyme secretion but not of bile lipid secretion suggests that the lysosomal activity is not directly involved in the bile secretion mechanism. These data point up the risks of using chlorpromazine therapy in association with a diet high in fat and cholesterol.     

Phospholipase A2 activity of rat stomach

Phospholipase A activity in rat stomach wall and in gastric content was studied using [1-14C]dioleoylphosphatidylcholine as substrate. The optimum activity of the stomach wall was found to take place at pH 7.0. During optimal phospholipase action about 40% of the [1-14C]oleic acid released was due to an active intracellular lysophospholipase. The gastric phospholipase required 5 mM Ca2+ for full activity and is inhibited by EDTA. It specifically hydrolyzed the sn-2 position of the phospholipid molecule. The enzyme was heat labile and inactivated by acidification at pH 3.0. The gastric content enzyme had a lower specific activity and an optimum pH of 8.0. It was heat stable and was not inactivated by acidification. These results indicate that gastric content phospholipase A is of pancreatic origin, via a duodenal reflux. By ligating the stomach we were able to further confirm that the gastric wall phospholipase was different from that of the gastric content. It originated from the stomach mucosa. Subcellular fractionation suggests that the gastric phospholipase A2 is essentially bound to the plasma membrane. About 6% of the activity was found to be soluble. Biopsies of human gastric mucosa displayed a phospholipase A activity which had similar properties to that of rat gastric enzyme. The physiological function of this enzyme is discussed in terms of prostaglandin synthesis via the release of arachidonic acid.     

Sequence diversity of pistil S-proteins associated with gametophytic self-incompatibility in Nicotiana alata

Summary In order to study the extent and nature of differences among various S-allele-associated proteins in N. alata, we carried out comparative studies of seven such proteins. We first isolated and sequenced cDNA clones for the S_z-, S_F11-, S₁-, and S_a-alleles, and then we compared the deduced amino acid sequences both of these four S-proteins and of three previously published S₂-, S₃-, and S₆-proteins. This comparison revealed (1) an average homology of 53.8% among the seven proteins and (2) two homology classes, with S_z and S_F11 in one class and S₁, S₂, S₃, and S₆ in the other class. There are 60 conserved residues, including 9 cysteines. Of the 144 variable residues, 50 were identified as hypervariable based on a calculation of their Similarity Indices. Although conserved, variable, and hypervariable residues are dispersed throughout the protein, some are clustered to form five conserved, five hypervariable, and a number of variable regions. Those variable sites which contain residues conserved within one class of S-proteins but different between classes might provide a clue to the evolutionary relationship of these two classes of S-proteins. The hypervariable residues, which account for sequence variability, may contribute to allelic specificity.     

Detection of a non-structural protein of M r 11 000 encoded by the virion DNA of maize streak virus

A polypeptide of approximately 11 000 daltons (11 kDa protein) encoded by an open reading frame (10.9 ORF) from the virion sense of maize streak virus (MSV) DNA has been detected among the products of in vitro translation reactions programmed with RNA from infected maize plants and also in total protein extracts from infected leaves. The 11 kDa protein has not been detected in virions and is therefore proposed to have a nonstructural role.Viral DNA with an additional in-frame translation stop codon in the 10.9 ORF was not infectious when transmitted to maize plants via Agrobacterium tumefaciens agroinfection, suggesting that the 10.9 ORF may be essential for virus function. Computer comparison data show that equivalent ORFs in wheat dwarf virus (WDV) and digitaria streak virus (DSV) have some sequences in common with the 10.9 ORF of MSV. Further-more, the absence of similar sequences in geminiviruses which infect dicotyledonous plants suggests that the 11 kDa protein and its putative homologs in WDV and DSV have a function necessary only for those geminiviruses which infect the Gramineae.The significance of the 11 kDa protein in relation to expression of the virion sense DNA of MSV is discussed.     

Stability of Bradyrhizobium japonicum Inoculants after Introduction into Soil

Bradyrhizobium japonicum USDA 125-Sp, USDA 138, and USDA 138-Sm had been used as inoculants for soybean (Glycine max (L.) Merr.) in soils previously free of B. japonicum. At 8 to 13 years after their release, these strains were reisolated from soil samples. A total of 115 isolates were obtained through nodules, and seven colonies were obtained directly by a serological method. The stability of the inoculants was confirmed by comparing the reisolated cultures with their respective parental strains which had been preserved by being lyophilized or stored on a yeast extract-mannitol agar slant at 4°C. Comparisons were made on morphological and serological characters, carbon compound utilization (8 tested), intrinsic antibiotic resistance (9 tested), and enzymatic activity (19 tested). Mucous and nonmucous isolates of serogroup 125 were analyzed for symbiotic effectiveness and restriction fragment hybridization with a DNA probe. Our data suggest that the B. japonicum inoculants have survived for up to 13 years in the soils without significant mutation except for two reisolates with a slightly increased kanamycin resistance level.     

Acute effects of filipin on the plasmic, hepatic, and biliary cholesterol of the rat

Twenty-one male Wistar rats, 13 weeks old, were fed ad libitum hyperlipidic diets (28% fats) loaded with cholesterol (1.2%) for 5 weeks. One group of 11 rats was fed saturated fats (diet group "S") and another group of 10 rats was fed polyunsaturated fats (diet group "PU"). On the day they were sacrificed 10 of the rats were injected intravenously with 1 mg of filipin. Contrary to the rats in diet group "PU," the rats in diet group "S" treated with filipin presented certain characteristics that were not found in the nontreated group: They provided evidence of biliary cholestasis accompanied by a decline in the level of secretion of bile salts and phospholipids into bile. The concentrations of both free and esterified cholesterol in plasma fell and the amount of (esterified) hepatic cholesterol rose, although there was no change due to the filipin in the amounts of hepatic phospholipids. Explanatory hypotheses for these phenomena were considered, first, at the site of plasma membranes where filipin binds selectively to the cholesterol in the membrane, causing a disruption which probably disturbs the absorbance of circulating lipoproteins, especially that of hepatocyte cells, particularly in diet group "PU." Second, the effects of filipin on subcellular membranes seem to disturb the secretion of lipids and lipoproteins into bile and plasma, especially in diet group "S." Last, at the intracellular level, filipin appears to have a blocking effect on the organelles involved in biliary lipid secretion. The activity of certain enzymes such as cholesterol esterase may also be blocked, particularly in diet group "S," which would explain the accumulation of esterified cholesterol in liver.