Small-subunit (SSU) rRNA gene sequences were obtained by PCR from 12 Blastocystis isolates from humans, rats, and reptiles for which elongation factor 1␣ (EF-1␣) gene sequences are already available. These new sequences were analyzed by the Bayesian method in a broad phylogeny including, for the first time, all Blastocystis sequences available in the databases. Phylogenetic trees identified seven well-resolved groups plus several discrete lineages that could represent newly defined clades. Comparative analysis of SSU rRNA-and EF-1␣-based trees obtained by maximum-likelihood methods from a restricted sampling (13 isolates) revealed overall agreement between the two phylogenies. In spite of their morphological similarity, sequence divergence among Blastocystis isolates reflected considerable genetic diversity that could be correlated with the existence of potentially >12 different species within the genus. Based on this analysis and previous PCR-based genotype classification data, six of these major groups might consist of Blastocystis isolates from both humans and other animal hosts, confirming the low host specificity of Blastocystis. Our results also strongly suggest the existence of numerous zoonotic isolates with frequent animal-to-human and human-to-animal transmissions and of a large potential reservoir in animals for infections in humans.

Objective-Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central player in the regulation of cholesterol homeostasis, increasing the low-density lipoprotein (LDL) receptor degradation. Our study aimed at exploring the pathogenic consequences in vivo and in vitro of a PCSK9 prodomain mutation found in a family with hypobetalipoproteinemia (FHBL). Methods and Results-A white 49-year-old diabetic man had profound FBHL (LDLC: 16 mg/dL) whereas his daughter and sister displayed a milder phenotype (LDLC 44 mg/dL and 57 mg/dL, respectively), all otherwise healthy with a normal liver function. A monoallelic PCSK9 double-mutant R104C/V114A cosegregated with FBHL, with no mutation found at other FHBL-causing loci. A dose-effect was also found in FBHL relatives for plasma APOB and PCSK9 (very-low to undetectable in proband, Ϸ50% decreased in sister and daughter) and LDL catabolic rate (256% and 88% increased in proband and daughter). Transient transfection in hepatocytes showed severely impaired processing and secretion of the double mutant which acted as a dominant negative over secretion of wild-type PCSK9. Conclusion-These results show that heterozygous PCSK9 missense mutations may associate with profound hypobetalipoproteinemia and constitute the first direct evidence in human that decrease of plasma LDLC concentrations associated to PCSK9 LOF mutations are attributable to an increased clearance rate of LDL. Key Words: PCSK9 Ⅲ LDL Ⅲ mutation Ⅲ hypobetalipoproteinemia H ypobetalipoproteinemia (HBL) refers to a heterogeneous group of monogenic disorders characterized by very low plasma concentrations of low-density lipoprotein cholesterol (LDLC) and apolipoprotein B (apoB) (ie, Ͻ5 percentile of the distribution in the population; for review see 1 ). HBL includes 3 inherited disorders: (1) familial hypobetalipoproteinemia (FHBL; OMIM 107730), (2) abetalipoproteinemia (ABL; OMIM 200100), and (3) chylomicron retention disease (CRD; OMIM 246700). The frequency of subjects with heterozygous FHBL has been estimated to be 1:500/1:1000. 2 FHBL heterozygotes are often asymptomatic or express mild clinical manifestations such as fatty liver disease and intestinal fat malabsorption. 3 FHBL are often caused by APOB gene mutations. 1,4 However, a substantial number of FHBL (varying to 36% 1 to 56% 5 in the literature) do not harbor apoB mutations.In the last 5 years, proprotein convertase subtilisin/kexin 9 (PCSK9) has emerged as a crucial modulator of cholesterol metabolism. 6 PCSK9 is primarily expressed in the liver and the intestine. PCSK9 inhibits the LDL receptor (LDLR) pathway in a posttranscriptional manner. 6 In human, PCSK9 was initially reported as the third gene causing autosomal dominant hypercholesterolemia, in addition to LDLR and APOB mutations. 7 Indeed, PCSK9 gain-of-function (GOF) mutations lead to increased plasma LDLC levels and premature atherosclerosis. 7,8 In contrast, PCSK9 loss-of-function (LOF) mutations are associated with low LDLC levels and protection against coronary diseases. 9,10 To dat...

The iron-and manganese-containing superoxide dismutases (Fe/Mn-SOD) share the same chemical function and spatial structure but can be distinguished according to their modes of oligomerization and their metal ion specificity. They appear as homodimers or homotetramers and usually require a specific metal for activity. On the basis of 261 aligned SOD sequences and 12 superimposed x-ray structures, two phenetic trees were constructed, one sequence-based and the other structurebased. Their comparison reveals the imperfect correlation of sequence and structural changes; hyperthermophilicity requires the largest sequence alterations, whereas dimer/tetramer and manganese/iron specificities are induced by the most sizable structural differences within the monomers. A systematic investigation of sequence and structure characteristics conserved in all aligned SOD sequences or in subsets sharing common oligomeric and/or metal specificities was performed. Several residues were identified as guaranteeing the common function and dimeric conformation, others as determining the tetramer formation, and yet others as potentially responsible for metal specificity. Some form cation-interactions between an aromatic ring and a fully or partially positively charged group, suggesting that these interactions play a significant role in the structure and function of SOD enzymes. Dimer/tetramer-and iron/manganese-specific fingerprints were derived from the set of conserved residues; they can be used to propose selected residue substitutions in view of the experimental validation of our in silico derived hypotheses.

Metalloenzymes such as the superoxide dismutases (SODs) form part of a defense mechanism that helps protect obligate and facultative aerobic organisms from oxygen toxicity and damage. Here, we report the presence in the trypanosomatid genomes of four SOD genes: soda, sodb1, sodb2, and a newly identified sodc. All four genes of Trypanosoma brucei have been cloned (Tbsods), sequenced, and overexpressed in Escherichia coli and shown to encode active dimeric FeSOD isozymes. Homology modeling of the structures of all four enzymes using available X-ray crystal structures of homologs showed that the four TbSOD structures were nearly identical. Subcellular localization using GFP-fusion proteins in procyclic insect trypomastigotes shows that TbSODB1 is mainly cytosolic, with a minor glycosomal component, TbSODB2 is mainly glycosomal with some activity in the cytosol, and TbSODA and TbSODC are both mitochondrial isozymes. Phylogenetic studies of all available trypanosomatid SODs and 106 dimeric FeSODs and closely related cambialistic dimeric SOD sequences suggest that the trypanosomatid SODs have all been acquired by more than one event of horizontal gene transfer, followed by events of gene duplication.

Trichomonads closely related to the bovid parasite Tritrichomonas foetus were identified in the bronchoalveolar lavage sample from a patient with AIDS in association with Pneumocystis pneumonia. This human case of T. foetus-like infection emphasizes the zoonotic potential of trichomonads, although the existence of a human-host-adapted T. foetus strain cannot be excluded. CASE REPORTA 54-year-old woman was admitted to the intensive care unit of Mantes-la-Jolie hospital, presenting with fever, severe fatigue, and dyspnea with polypnea that had been increasing over the previous month. The patient was known to be seropositive for human immunodeficiency virus, which was revealed 3 years previously following pneumocystosis. At that time, an antiretroviral therapy was proposed but the patient denied the diagnosis. Since the patient had no history of blood transfusion or injection of illicit drugs, transmission by unprotected sex remained the presumed mode of human immunodeficiency virus contamination. In addition, the patient was overweight and had type 2 diabetes. Hemodynamic parameters were within normal limits. Blood gases were 52 mm Hg O 2 and 36 mm Hg CO 2 . A chest X-ray showed bilateral interstitial syndrome and several alveolar opacities. Bronchoalveolar lavage (BAL) was performed by fiber optic bronchoscopy and confirmed pneumocystosis. Interestingly, microscopic examination of a MayGrünwald-Giemsa-stained cytospin slide revealed numerous cells assumed to be trichomonad organisms, mixed with Pneumocystis parasites. The BAL sample was therefore immediately frozen for further analysis. Nasal oxygenation, intravenous injection of trimethoprim-sulfamethoxazole and methylprednisolone hemisuccinate, and subcutaneous injection of insulin were started, and recovery was rapid. On day 4, the patient was transferred to the Department of Infectious Diseases of the same institution where the investigations were completed. The CD4 cell count was 60 per mm 3 , with a CD4/CD8 ratio of 0.06.Plasma viral load was 5.34 log 10 copies/ml. Hypercalcemia with hyperparathyroidism was also noticed. The patient returned home 13 days after her hospitalization with trimethoprim-sulfamethoxazole per os. Antiretroviral therapy and exploration of hyperparathyroidism were scheduled.

Tritrichomonas foetus is the causative agent of bovine trichomonosis. This protozoan is found in the preputial cavity of bulls and is transmitted to cows during coitus. Currently, the diagnosis of this parasite is based on microscopic examination of preputial washings or scrapings, but it was recently recognized that other trichomonads similar in size, shape, and motility to T. foetus can be present in preputial samples. Despite the serious consequences of an incorrect diagnosis for bovine trichomonosis, the precise speciation of these other trichomonads has remained uncertain. Here, a total of 12 non-T. foetus isolates were microscopically examined. On the basis of morphological criteria, seven of these isolates were identified as Tetratrichomonas sp., whereas four other isolates coincided with the description of Pentatrichomonas hominis. In the last isolate, a third non-T. foetus species was identified as belonging to the genera Pseudotrichomonas or Monocercomonas: the first time that species of either of these genera have been reported in preputial samples. To confirm these data, small subunit rRNA gene sequences were obtained by PCR from the 12 trichomonad isolates. These new sequences were analysed in a broad phylogeny including 72 other parabasalid sequences. From our phylogenetic trees, we confirmed the taxonomic status of non-T. foetus organisms isolated from preputial samples (Tetratrichomonas, Pentatrichomonas, and Pseudotrichomonas) and suggested the existence of two Tetratrichomonas species, despite their morphological similarity. The route of transmission of the non-T. foetus organisms identified in the bovine preputial cavity is discussed and we confirm that the PCR assay using the previously described T. foetus-specific primers TFR3 and TFR4 could be a useful alternative method for the diagnosis of bovine trichomonosis.

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This article is available online at http://www.jlr.org frequency of 1.3% in the cohort. Therefore, even though LDLR mutations are the major cause of ADH with a large mutation spectrum, APOE variants were found to be significantly associated with the disease. Furthermore, using structural analysis and modeling, the identifi ed APOE sequence changes were predicted to impact protein function. Abstract Autosomal dominant hypercholesterolemia (ADH) is a human disorder characterized phenotypically by isolated high-cholesterol levels. Mutations in the low density lipoprotein receptor ( LDLR ), APOB , and proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) genes are well known to be associated with the disease. To characterize the genetic background associated with ADH in France, the three ADHassociated genes were sequenced in a cohort of 120 children and 109 adult patients. Fifty-one percent of the cohort had a possible deleterious variant in LDLR , 3.1% in APOB , and 1.7% in PCSK9 . We identifi ed 18 new variants in LDLR and 2 in PCSK9 . Three LDLR variants, including two newly identifi ed, were studied by minigene reporter assay confi rming the predicted effects on splicing. Additionally, as recently an in-frame deletion in the APOE gene was found to be linked to ADH, the sequencing of this latter gene was performed in patients without a deleterious variant in the three former genes. An APOE variant was identifi ed in three patients with isolated severe hypercholesterolemia giving a