A genome-informed higher rank classification of the biotechnologically important fungal subphylum Saccharomycotina.

The subphylum Saccharomycotina is a lineage in the fungal phylum Ascomycota that exhibits levels of genomic diversity similar to those of plants and animals. The Saccharomycotina consist of more than 1 200 known species currently divided into 16 families, one order, and one class. Species in this subphylum are ecologically and metabolically diverse and include important opportunistic human pathogens, as well as species important in biotechnological applications. Many traits of biotechnological interest are found in closely related species and often restricted to single phylogenetic clades. However, the biotechnological potential of most yeast species remains unexplored. Although the subphylum Saccharomycotina has much higher rates of genome sequence evolution than its sister subphylum, Pezizomycotina, it contains only one class compared to the 16 classes in Pezizomycotina. The third subphylum of Ascomycota, the Taphrinomycotina, consists of six classes and has approximately 10 times fewer species than the Saccharomycotina. These data indicate that the current classification of all these yeasts into a single class and a single order is an underappreciation of their diversity. Our previous genome-scale phylogenetic analyses showed that the Saccharomycotina contains 12 major and robustly supported phylogenetic clades; seven of these are current families (Lipomycetaceae, Trigonopsidaceae, Alloascoideaceae, Pichiaceae, Phaffomycetaceae, Saccharomycodaceae, and Saccharomycetaceae), one comprises two current families (Dipodascaceae and Trichomonascaceae), one represents the genus Sporopachydermia, and three represent lineages that differ in their translation of the CUG codon (CUG-Ala, CUG-Ser1, and CUG-Ser2). Using these analyses in combination with relative evolutionary divergence and genome content analyses, we propose an updated classification for the Saccharomycotina, including seven classes and 12 orders that can be diagnosed by genome content. This updated classification is consistent with the high levels of genomic diversity within this subphylum and is necessary to make the higher rank classification of the Saccharomycotina more comparable to that of other fungi, as well as to communicate efficiently on lineages that are not yet formally named. Taxonomic novelties: New classes: Alloascoideomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Dipodascomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Lipomycetes M. Groenew., Hittinger, Opulente, A. Rokas, Pichiomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Sporopachydermiomycetes M. Groenew., Hittinger, Opulente & A. Rokas, Trigonopsidomycetes M. Groenew., Hittinger, Opulente & A. Rokas. New orders: Alloascoideomycetes: Alloascoideales M. Groenew., Hittinger, Opulente & A. Rokas; Dipodascomycetes: Dipodascales M. Groenew., Hittinger, Opulente & A. Rokas; Lipomycetes: Lipomycetales M. Groenew., Hittinger, Opulente & A. Rokas; Pichiomycetes: Alaninales M. Groenew., Hittinger, Opulente & A. Rokas, Pichiales M. Groenew., Hittinger, Opulente & A. Rokas, Serinales M. Groenew., Hittinger, Opulente & A. Rokas; Saccharomycetes: Phaffomycetales M. Groenew., Hittinger, Opulente & A. Rokas, Saccharomycodales M. Groenew., Hittinger, Opulente & A. Rokas; Sporopachydermiomycetes: Sporopachydermiales M. Groenew., Hittinger, Opulente & A. Rokas; Trigonopsidomycetes: Trigonopsidales M. Groenew., Hittinger, Opulente & A. Rokas. New families: Alaninales: Pachysolenaceae M. Groenew., Hittinger, Opulente & A. Rokas; Pichiales: Pichiaceae M. Groenew., Hittinger, Opulente & A. Rokas; Sporopachydermiales: Sporopachydermiaceae M. Groenew., Hittinger, Opulente & A. Rokas. Citation: Groenewald M, Hittinger CT, Bensch K, Opulente DA, Shen X-X, Li Y, Liu C, LaBella AL, Zhou X, Limtong S, Jindamorakot S, Gonçalves P, Robert V, Wolfe KH, Rosa CA, Boekhout T, Cadez N, Péter G, Sampaio JP, Lachance M-A, Yurkov AM, Daniel H-M, Takashima M, Boundy-Mills K, Libkind D, Aoki K, Sugita T, Rokas A (2023). A genome-informed higher rank classification of the biotechnologically important fungal subphylum Saccharomycotina. Studies in Mycology 105: 1-22. doi: 10.3114/sim.2023.105.01 This study is dedicated to the memory of Cletus P. Kurtzman (1938-2017), a pioneer of yeast taxonomy.

The cytokine profile of women with severe anxiety and depression during pregnancy.

BACKGROUND: Controversial findings regarding the association between pro-inflammatory cytokines and depression have been reported in pregnant subjects. Scarce data about anxiety and its relationships with cytokines are available in pregnant women. To understand the association between anxiety and cytokines during pregnancy, we conducted the present study in women with or without depression. METHODS: Women exhibiting severe depression (SD) and severe anxiety (SA) during the 3rd trimester of pregnancy (n = 139) and control subjects exhibiting neither depression nor anxiety (n = 40) were assessed through the Hamilton Depression Rating Scale (HDRS) and the Hamilton Anxiety Rating Scale (HARS). Serum cytokines were measured by a multiplex bead-based assay. Correlation tests were used to analyze the data and comparisons between groups were performed. A general linear model of analysis of variance was constructed using the group as a dependent variable, interleukin concentrations as independent variables, and HDRS/HARS scores and gestational weeks as covariables. RESULTS: The highest levels of Th1- (IL-6, TNF-α, IL-2, IFN-γ), Th17- (IL-17A, IL-22), and Th2- (IL-9, IL-10, and IL-13) related cytokines were observed in women with SD + SA. The SA group showed higher concentrations of Th1- (IL-6, TNF-α, IL-2, IFN-γ) and Th2- (IL-4, and IL-10) related cytokines than the controls. Positive correlations were found between HDRS and IL-2, IL-6, and TNF-α in the SA group (p < 0.03), and between HDRS and Th1- (IL-2, IL-6, TNF-α), Th2- (IL-9, IL-10, IL-13) and Th17- (IL-17A) cytokines (p < 0.05) in the SD + SA group. After controlling the correlation analysis by gestational weeks, the correlations that remained significant were: HDRS and IL-2, IL-6, IL-9, and IL-17A in the SD + SA group (p < 0.03). HARS scores correlated with IL-17A in the SA group and with IL-17A, IL-17F, and IL-2 in the SD + SA group (p < 0.02). The linear model of analysis of variance showed that HDRS and HARS scores influenced cytokine concentrations; only IL-6 and TNF-α could be explained by the group. CONCLUSIONS: We found that the cytokine profiles differ when comparing pregnant subjects exhibiting SA with comorbid SD against those showing only SA without depression.

Knee osteoarthritis pain following medial meniscectomy in the nonhuman primate.

OBJECTIVE: A number of promising compounds developed for osteoarthritic pain have failed to demonstrate clinical efficacy. To enhance preclinical translational research for osteoarthritis, a model of knee osteoarthritis pain was developed in Macaca fascicularis and the effects of two distinct pharmacological classes of drugs were tested on pain-related behavior. DESIGN: Behavioral assessments were developed specifically for the macaque. Baseline knee pressure threshold and weight bearing were assessed prior to a unilateral medial meniscectomy (MMx). Fifteen days following MMx, macaques underwent a once daily exercise regimen for 36 days. Sixty-seven days following MMx, macaques were assigned to one of three treatment groups (n = 3/group), either non-steroidal anti-inflammatory drug (NSAID) diclofenac, NK1 receptor antagonist aprepitant or vehicle, and treated for 5 days. Animals were tested 3-4 h after p.o. dosing and testing was performed blinded. Treatment utilized a crossover design-each animal received all treatments-and a 9-day washout period was utilized between treatments. RESULTS: Vehicle-treated macaques consistently demonstrated decreased ipsilateral pressure threshold ("hyperalgesia") and decreased weight bearing. While diclofenac increased weight bearing and pressure threshold, full attenuation of pain was not obtained. No significant improvement of either knee pressure or weight bearing was observed with aprepitant. CONCLUSIONS: Unilateral MMx in the macaque evoked pain-related behaviors and knee joint pathology reminiscent of osteoarthritis. The behavioral endpoints were sensitive to NSAID treatment but not sensitive to NK1 receptor block, which parallel clinical findings. The current macaque osteoarthritis model could be used to test potential treatments for osteoarthritis pain.

Phylogeny of yeasts and related filamentous fungi within Pucciniomycotina determined from multigene sequence analyses.

In addition to rusts, the subphylum Pucciniomycotina (Basidiomycota) includes a large number of unicellular or dimorphic fungi which are usually studied as yeasts. Ribosomal DNA sequence analyses have shown that the current taxonomic system of the pucciniomycetous yeasts which is based on phenotypic criteria is not concordant with the molecular phylogeny and many genera are polyphyletic. Here we inferred the molecular phylogeny of 184 pucciniomycetous yeast species and related filamentous fungi using maximum likelihood, maximum parsimony and Bayesian inference analyses based on the sequences of seven genes, including the small subunit ribosomal DNA (rDNA), the large subunit rDNA D1/D2 domains, the internal transcribed spacer regions (ITS 1 and 2) of rDNA including the 5.8S rDNA gene; the nuclear protein-coding genes of the two subunits of DNA polymerase II (RPB1 and RPB2) and the translation elongation factor 1-α (TEF1); and the mitochondrial gene cytochrome b (CYTB). A total of 33 monophyletic clades and 18 single species lineages were recognised among the pucciniomycetous yeasts employed, which belonged to four major lineages corresponding to Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes and Mixiomycetes. These lineages remained independent from the classes Atractiellomycetes, Classiculomycetes, Pucciniomycetes and Tritirachiomycetes formed by filamentous taxa in Pucciniomycotina. An updated taxonomic system of pucciniomycetous yeasts implementing the 'One fungus = One name' principle will be proposed based on the phylogenetic framework presented here.

Frequency of sinus disease in normal subjects and patients with benign paroxysmal positional vertigo.

BACKGROUND/AIMS: To determine if patients with benign paroxysmal positional vertigo (BPPV) have a higher frequency of rhinosinusitis than people with normal vestibular function. METHODS: The subjects were 52 patients with BPPV and 46 normal people. Every subject had a sinus CT scan, a blood draw for IgE and a rhinologic examination by an otolaryngologist. RESULTS: The frequency of rhinosinusitis based on physician diagnosis was 49% and based on CT scan findings 59%. This difference approached significance (p = 0.08). The observed frequency of rhinosinusitis was higher than predicted by survey data about the southern US region. The data trended toward higher prevalence of rhinosinusitis (by physician diagnosis) in the BPPV patients versus controls (58 vs. 39%, p = 0.06). CONCLUSION: BPPV patients have a higher frequency of sinus disease compared to people with normal vestibular systems, perhaps due to age differences, but physiologic factors may also be involved. The higher frequency of rhinosinusitis in this geographical area than reported rates based on survey data raises concerns about the usefulness of questionnaire data for estimating population prevalence.

Inter- and intra-rater reliability in fibroptic endoscopic evaluation of swallowing.

Fibroptic endoscopic evaluation of swallowing (FEES) is a useful way for dentists to evaluate oropharyngeal dysfunction. However, no study has paid attention to inter- and intra-rater reliability of FEES evaluation about oropharyngeal dysfunction. The purpose of this study is to verify whether dentist who trained and experienced for evaluation of dysphagia could diagnose oropharyngeal function with FEES. Nine dentists independently evaluated FEES images of 10 cases four times each. At first, evaluators performed the first evaluation without consulting the evaluative criteria. Subsequently, evaluators independently re-evaluated at 1-week intervals for three consecutive weeks, consulting the evaluative criteria. And then, inter- and intra-rater reliability was calculated. Cohen's Kappa was used to assess reliability. The results found that overall inter-rater reliability was 0·35±0·04 (first evaluation), 0·45±0·05 (s), 0·44±0·05 (third) and 0·46±0·04 (fourth). Most of inter-rater reliability related to aspiration was moderate to high, but lower for categories that evaluated timing of swallowing and mastication. In contrast, intra-rater reliability was moderate to high for overall categories, at 0·53±0·04 (first vs. second evaluation), 0·55±0·04 (first vs. third), 0·53±0·04 (first vs. fourth), 0·55±0·03 (second vs. third), 0·60±0·03 (second vs. fourth) and 0·78±0·03 (third vs. fourth). FEES is reliable for experienced dentists to diagnose oropharyngeal function. Moreover, repeated evaluation with the aids of evaluative criteria is useful to improve the reliability of FEES.

Application of acidic thermal treatment for one- and two-stage anaerobic digestion of sewage sludge.

The effectiveness of acidic thermal treatment (ATT) was examined in a 106-day continuous experiment, when applied to one- or two-stage anaerobic digestion of sewage sludge (4.3% TS). The ATT was performed at 170 °C and pH 5 for 1 hour (sulfuric acid for lowering pH). The one-stage process was mesophilic at 20 days hydraulic retention time (HRT), and incorporated the ATT as pre-treatment. The two-stage process consisted of a thermophilic digester at 5 days HRT and a mesophilic digester at 15 days HRT, and incorporated the ATT as interstage-treatment. On average, VSS reduction was 48.7% for the one-stage control, 65.8% for the one-stage ATT, 52.7% for the two-stage control and 67.6% for the two-stage ATT. Therefore, VSS reduction was increased by 15-17%, when the ATT was combined in both one- and two-stage processes. In addition, the dewaterability of digested sludge was remarkably improved, and phosphate release was enhanced. On the other hand, total methane production did not differ significantly, and color generation was noted in the digested sludge solutions with the ATT. In conclusion, the anaerobic digestion with ATT can be an attractive alternative for sludge reduction, handling, and phosphorus recovery.

Molecular characterization of the bovine chromodomain Y-like genes.

The human chromodomain protein, Y-like (CDYL) gene family consists of three members, one on the Y chromosome (CDY) and two on autosomes (CDYL and CDYL2). Studies in the human and mouse showed that genes in the CDYL family are abundantly expressed in testis and play an important role in spermatogenesis. In this study, we have characterized the bovine CDYL (bCDYL) and CDYL2 (bCDYL2) genes. We found that bCDYL and bCDYL2 are very similar to the human orthologues at both mRNA (79% and 85%) and protein (89% and 93%) levels. However, the similarity between the bCDYL and bCDYL2 proteins is low (41%). The bCDYL gene is composed of nine exons, and the bCDYL2 has seven exons. The bCDYL and bCDYL2 genes were mapped by radiation hybrid mapping to bovine chromosomes (BTA) 24 and 18 respectively. The bCDYL gene has four transcript variants that produce four protein isoforms. RT-PCR expression analysis in 12 bovine tissues showed that bCDYL variant 2 was expressed in the testis only, bCDYL variants 1, 3 and 4 were expressed predominantly in the testis and at very low or undetectable levels in the remaining tissues and bCDYL2 was expressed ubiquitously. Examination of bovine testis with in situ hybridization revealed that the bCDYL and bCDYL2 transcripts were found mainly in spermatids, though the amounts of transcripts varied among genes/variants. In addition, antisense transcripts were detected in bCDYL variants 2/3 and 4, as well as in the bCDYL2 gene.

Partial characterization of the antigen recognized by a monoclonal antibody to Ascaris suum ovary extracts.

A monoclonal antibody produced against ovary extracts from the worm Ascaris suum showed immunoreactivity against granules in the rachis and oocytes, the inner layer of the eggshell and the middle layer of some egg, but not against either ovary wall or uterus wall. Furthermore, the same antigens were detected on the body surface of migrated larva in guinea pig lung, whereas none were detected in adult male worm or adult female worm, except for the female reproductive organs. The ovary extracts were passed through an affinity column and the eluted fractions analyzed by SDS-PAGE, Western blotting and native-PAGE. Western blotting after SDS-PAGE detected chemiluminescence primarily as three bands of about 70, 78 and 90 kDa. However, Western blotting after native-PAGE of the partially purified ovary extracts demonstrated only one band at a position of about 230 kDa. LC-nanoESI-MS/MS analysis of protein band gel slices from silver-stained SDS-PAGE revealed one peptide sequence "ILVGLIGTNR", that matched only the hypothetical protein F14D2.8 of Caenorhabditis elegans (gi/7499081).

Effects of genotypic differences in CYP2C19 status on cure rates for Helicobacter pylori infection by dual therapy with rabeprazole plus amoxicillin.

Rabeprazole is a potent proton pump inhibitor and is mainly reduced to thioether rabeprazole by a non-enzymatic pathway and partially metabolized to demethylated rabeprazole by CYP2C19 in the liver. We intended to determine a cure rate for Helicobacter pylori infection by dual rabeprazole/amoxicillin therapy in relation to CYP2C19 genotype status prospectively. Ninety-seven patients with gastritis and H. pylori infection completed the dual therapy with 10 mg of rabeprazole bid and 500 mg of amoxicillin tid for 2 weeks. At 1 month after treatment, cure of H. pylori infection was assessed on the basis of histology, a rapid urease test, culture, polymerase chain reaction (PCR), and 13C-urea breath test. CYP2C19 genotype status was determined by a PCR-restriction fragment length polymorphism method. Of the 97 patients, 33 were homozygous extensive metabolizers (homEM), 48 were heterozygous extensive metabolizers (hetEM), and 16 were poor metabolizers (PM). Cure of H. pylori infection was achieved in 79 of the 97 patients (81.4%, 95%CI = 71.9-88.7). Significant differences in cure rates among the homEM, hetEM, and PM groups were observed; 60.6% (95%CI = 42.1-77.3), 91.7% (95%CI = 80.0-97.7), and 93.8% (95%CI = 69.8-99.8), respectively (P = 0.0007). Twelve patients without cure after initial treatment (10 homEMs and 2 hetEMs) were successfully retreated with rabeprazole 10 mg q.i.d. and amoxicillin 500 mg q.i.d. for 2 weeks. The cure rates for H. pylori infection by dual rabeprazole/amoxicillin therapy depended on the CYP2C19 genotype status. This dual therapy appears to be effective for hetEM and PM patients. However, high dose dual rabeprazole/amoxicillin therapy was effective even for homEM patients. Therefore, the genotyping test of CYP2C19 appears to be a clinically useful tool for the optimal dual treatment with rabeprazole plus amoxicillin.

Cure of refractory duodenal ulcer and infection caused by Helicobacter pylori by high doses of omeprazole and amoxicillin in a homozygous CYP2C19 extensive metabolizer patient.

A 53-year old female patient with duodenal ulcer and Helicobacter pylori infection was treated three times with a proton pump inhibitor-based triple therapy, such as lansoprazole-clarithromycin-amoxicillin (INN, amoxicilline) and lansoprazole-minocycline-cefaclor. However, the H pylori infection was not cured. A culture test revealed that her infection was a clarithromycin-resistant but amoxicillin-sensitive strain of H pylori. Moreover, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed that she was a homozygous extensive metabolizer of cytochrome P450 (CYP) 2C19 (wt/wt). The usual dose of the proton pump inhibitor was therefore assumed to be insufficient for her and then she was treated with a high dose of omeprazole (120 mg/day) and amoxicillin (2,250 mg/day) for 2 weeks. The H pylori infection and the ulcer lesion were then cured. One of the factors associated with success or failure of cure of H pylori infection by the proton pump inhibitor-based triple therapy appeared to be CYP2C19 genotype status. Dual treatment with a sufficient dose of a proton pump inhibitor plus amoxicillin could cure H pylori infection even after the failure to cure H pylori infection by a usual proton pump inhibitor-based triple therapy in patients with the wt/wt homozygous extensive metabolizer genotype of CYP2C19.

Effects of clarithromycin on the metabolism of omeprazole in relation to CYP2C19 genotype status in humans.

BACKGROUND AND PURPOSE: A triple therapy with omeprazole, amoxicillin (INN, amoxicilline), and clarithromycin is widely used for the eradication of Helicobacter pylori. Omeprazole and clarithromycin are metabolized by CYP2C19 and CYP3A4. This study aimed to elucidate whether clarithromycin affects the metabolism of omeprazole. METHODS: After administration of placebo or 400 mg clarithromycin twice a day for 3 days, 20 mg omeprazole and placebo or 400 mg clarithromycin were administered to 21 healthy volunteers. Plasma concentrations of omeprazole and clarithromycin and their metabolites were determined before and 1, 2, 3, 5, 7, 10, and 24 hours after dosing. CYP2C19 genotype status was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: Subjects were classified into three groups on the basis of PCR-RFLP analyses for CYP2C19: homozygous extensive metabolizer group (n = 6), heterozygous extensive metabolizer group (n = 11), and poor metabolizer group (n = 4). Mean area under the plasma concentration-time curves from 0 to 24 hours (AUC) of omeprazole in the homozygous extensive metabolizer, heterozygous extensive metabolizer, and poor metabolizer groups were significantly increased by clarithromycin from 383.9 to 813.1, from 1001.9 to 2110.4, and from 5589.7 to 13098.6 ng x h/mL, respectively. There were significant differences in the mean AUC values of clarithromycin among the three groups. CONCLUSION: Clarithromycin inhibits the metabolism of omeprazole. Drug interaction between clarithromycin and omeprazole may underlie high eradication rates achieved by triple therapy with omeprazole, amoxicillin, and clarithromycin.

CYP2C19 genotype status and effect of omeprazole on intragastric pH in humans.

OBJECTIVE: Omeprazole is metabolized by genetically determined S-mephenytoin 4'-hydroxylase (CYP2C19) in the liver. This study aimed to determine whether the effect of omeprazole on intragastric pH depends on CYP2C19 genotype status. METHODS: CYP2C19 genotype status for 2 mutations associated with the poor metabolizer phenotype was determined by a polymerase chain reaction-restriction fragment length polymorphism method in 16 healthy volunteers. Helicobacterpylori status was determined by serology and the [13C]urea breath test. After a single oral administration of 20 mg omeprazole or a placebo, intragastric pH values were recorded for 24 hours. Plasma levels of omeprazole and its 2 metabolites and gastrin were measured before and 1, 2, 3, 5, 7, 10, and 24 hours after administration. RESULTS: Fifteen of the 16 subjects were H pylori negative. Five of the 15 subjects were homozygous extensive metabolizers, 4 were heterozygous extensive metabolizers, and 6 were poor metabolizers. After omeprazole administration, significant differences in mean intragastric pH values and plasma levels of gastrin, omeprazole and its metabolites were observed among the 3 groups, whereas no significant differences in these parameters were observed with the placebo administration. CONCLUSIONS: The effect of omeprazole on intragastric pH significantly depends on CYP2C19 genotype status. The genotyping test of CYP2C19 may be useful for an optimal prescription of omeprazole.

Effect of genotypic differences in CYP2C19 on cure rates for Helicobacter pylori infection by triple therapy with a proton pump inhibitor, amoxicillin, and clarithromycin.

BACKGROUND: Proton pump inhibitors such as omeprazole and lansoprazole are mainly metabolized by CYP2C19 in the liver. The therapeutic effects of proton pump inhibitors are assumed to depend on CYP2C19 genotype status. OBJECTIVE: We investigated whether CYP2C19 genotype status was related to eradication rates of H pylori by triple proton pump inhibitor-clarithromycin-amoxicillin (INN, amoxicilline) therapy and attempted to establish a strategy for treatment after failure to eradicate H pylori. METHODS: A total of 261 patients infected with H pylori completed initial treatment with 20 mg of omeprazole or 30 mg of lansoprazole twice a day, 200 mg of clarithromycin three times a day, and 500 mg of amoxicillin three times a day for 1 week. CYP2C19 genotypes of patients were determined with polymerase chain reaction-restriction fragment length polymorphism analysis. Patients without eradication after initial treatment were retreated with 30 mg of lansoprazole four times daily and 500 mg of amoxicillin four times daily for 2 weeks. RESULTS: Eradication rates for H pylori were 72.7% (95% confidence interval, 64.4%-81.8%), 92.1% (confidence interval, 86.4%-97.3%), and 97.8% (confidence interval, 88.5%-99.9%) in the homozygous extensive, heterozygous extensive, and poor metabolizer groups, respectively. Thirty-four of 35 patients without eradication had an extensive metabolizer genotype of CYP2C19. Nineteen of those patients were infected with clarithromycin-resistant strains of H pylori. However, there were no amoxicillin-resistant strains of H pylori. Re-treatment of H pylori infection with dual high-dose lansoprazole-amoxicillin therapy succeeded in 30 of 31 patients with extensive metabolizer genotype of CYP2C19. CONCLUSION: The majority of patients without initial eradication of H pylori had an extensive metabolizer CYP2C19 genotype but were successfully re-treated with high doses of lansoprazole and an antibiotic to which H pylori was sensitive, such as amoxicillin, even when the patients were infected with clarithromycin-resistant strains of H pylori.

Down-regulation of ubiquitin gene expression during differentiation of human leukemia cells.

Ubiquitin, which is ligated covalently to target proteins for their acquisition of a variety of functions, is encoded by multiple unique genes in human cells: two distinct poly-ubiquitin genes with tandemly repeated sequences of 3 or 9 moieties and two mono-ubiquitin genes fused with small and large ribosomal proteins. We found that all classes of ubiquitin genes as well as the two genes encoding the ribosomal proteins S17 and L31 were expressed at abnormally high levels in various hematopoietic malignant tumor cells. In contrast, in vitro terminal differentiation of various immature leukemic cell lines, such as HL-60 promyelocytic leukemia cells and K562 erythroleukemia cells into monocytic, granulocytic and erythroid cells, induced by various agents was found to cause rapid and marked down-regulation of ubiquitin expression, irrespective of the cell type, direction of differentiation or type of signal. These findings suggest that the expressions of the multiple ubiquitin genes, coordinated with those of the ribosomal protein genes, are in a dynamic state during growth and differentiation of leukemia cells.

Different cytokines are required for induction and maintenance of the Th2 type response in DBA/2 mice resistant to infection with Leishmania major.

Experimental cutaneous leishmaniasis is a useful model in studying the mechanism regulating immune responses between T helper type 1 (Th1) and Th2. Mice susceptible to Leishmania major infection such as BALB/c (H-2(d)) are associated with the induction of the disease-promoting Th2 response, while the resistant mice such as DBA/2 (H-2(d)) develop the protective Th1 response. To understand the induction mechanism of Th1 and Th2 responses, it is necessary to establish an immunization scheme by which the induction of each Th response can be easily and experimentally controlled. Adjuvants are known to enhance the immune responses through the combined effect of several factors: prolonged release of antigen, migration of cells, mitogenic effect and so forth. When the genetically resistant DBA/2 mice were immunized twice with soluble leishmanial antigen (SLA), emulsified in incomplete Freund's adjuvant (IFA) before L. major inoculation, these mice mounted a Th2 cell response and suffered from progressive infection. While IL-4 and IL-13 were upregulated early after the infection in both healer and non-healer groups of mice, IL-5 and IL-10 were upregulated only in non-healer mice. From these results, IL-5 and IL-10 appear to have an important role, at least in the early phases of the infection, rather than IL-4 and IL-13 in establishing the disease-promoting Th2 response in leishmaniasis. Further, IL-9 was found to be expressed in both BALB/c and DBA/2 mice immunized with IFA/SLA. This cytokine may support the establishment of a Th2 response in these mice. Therefore it is suggested that Th2 cytokines play different roles between priming and maintaining the Th2 immune response after the infection.

A gene on SCA4 locus causes dominantly inherited pure cerebellar ataxia.

BACKGROUND: Several different genes or their loci have been identified for autosomal dominant cerebellar ataxia (ADCA). However, other types of ataxia remain unassigned. OBJECTIVE: To identify a new locus for ADCA. METHODS: Six Japanese families with ADCA with pure cerebellar syndrome (ADCA type III) were examined. These families had been molecularly excluded for spinocerebellar ataxia (SCA) types 1 through 3, 5 through 8, and 10. Clinical examination was undertaken, and a genome-wide linkage search was performed on 250 microsatellite DNA markers. RESULTS: Strong evidence for linkage was found with markers on human chromosome 16q, and haplotype and multipoint analyses further refined the gene locus in a 10.9-cM interval between D16S3089 and D16S515. Linkage disequilibrium was further found with the marker D16S3107 within the interval. The locus was exactly the candidate interval of SCA4, a rare form of ADCA clinically characterized by ataxia with sensory neuropathy and pyramidal tract signs. This would suggest that SCA4 and our ADCA type III are likely to be allelic disorders with different clinical features. CONCLUSION: The current study provides evidence that a gene on the SCA4 locus causes a pure cerebellar syndrome.

An inherited prion disease with a PrP P105L mutation: clinicopathologic and PrP heterogeneity.

OBJECTIVE: To clarify a clinical and neuropathologic phenotype of an inherited prion disease associated with a missense mutation at codon 105 in the prion protein (PrP) gene that was originally described as a variant of Gerstmann-Sträussler-Scheinker disease demonstrating spastic paraparesis. METHODS: Two siblings from a Japanese family are described. PrP gene analyses, neuropathologic studies with immunohistochemistry, and Western blot analysis of the PrP were performed. RESULTS: Both patients showed a missense (proline-->leucine) mutation at codon 105 and a methionine/valine polymorphism at codon 129 of the PrP gene. Clinically, Patient 1 presented with progressive spastic paraparesis, ataxia, and dementia. Patient 2, the sister of Patient 1, showed prominent action myoclonus and dementia. Neuropathologically, multiple PrP-positive amyloid plaques and diffuse PrP deposition in the deep cortical layers were found in the cerebral cortex with primarily frontal dominant atrophy in both patients. Tau-positive pathologic structures including neurofibrillary tangles, neuropil threads, and dystrophic neurites around the plaques were abundant in the brain of Patient 2. In contrast, the tau pathology was scarce in Patient 1. Western blot analysis of the brain showed different patterns of detergent-insoluble PrP fragments between the patients. CONCLUSIONS: Despite the identical codon 105 mutation and codon 129 polymorphism of the PrP gene, remarkable clinical and neuropathologic differences, and PrP heterogeneity were present between the affected siblings. The phenotypic variability might be related to PrP heterogeneity.

Haloperidol in large doses reduces the cataleptic response and increases noradrenaline metabolism in the brain of the rat.

The neurochemical basis for the clinical observation that some patients receiving a large dose of haloperidol exhibit no extrapyramidal side effects was investigated in rats. Haloperidol at doses of 1, 2.5, 5, 7.5 and 10 mg/kg (i.p.) caused a dose-dependent decrease in the duration of catalepsy. Haloperidol at a dose of 10 mg/kg induced catalepsy lasting for only 20% of that obtained with 1 mg/kg. Haloperidol decreased the content of noradrenaline in the frontal cortex and thalamus in a dose-dependent manner, while the content of 3-methoxy-4-hydroxyphenylglycol (MHPG) showed a dose-dependent increase in the same areas of the brain. Thus, there was an inverse relationship between the duration of catalepsy and the ratio of 3-methoxy-4-hydroxyphenylglycol to noradrenaline in the frontal cortex or thalamus. The concomitant administration of 20 mg/kg of phenoxybenzamine with 10 mg/kg of haloperidol induced a long-lasting catalepsy. The result may indicate that the increased metabolism of noradrenaline by large doses of haloperidol was not secondary to the blocking of dopaminergic receptors. In contrast, haloperidol caused a dose-dependent decrease in the content of homovanillic acid and 3,4-dihydroxyphenylacetic acid in the striatum and mesolimbic area. These results indicate that noradrenergic hyperfunction in the frontal cortex or thalamus induced by large doses of haloperidol may reduce the cataleptogenic effect of the drug via indirect stimulation of a dopaminoceptive neuron in the striatum or mesolimbic area.

Synthesis and neuroleptic activity of benzamides. Cis-N-(1-benzyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4-(methylamino)benzamide and related compounds.

Three series of benzamides of N,N-disubstituted ethylenediamines (linear alkane-1,2-diamines), 1-substituted 2-(aminomethyl)pyrrolidines, and 1-substituted 3-aminopyrrolidines (cyclic alkane-1,2-diamines) were designed and synthesized as potential neuroleptics. All target compounds were evaluated for their inhibitory effects on apomorphine-induced stereotyped behavior in rats, and a good correlation between structure and activity was found throughout the series. In the linear series (analogues of metoclopramide), introduction of a benzyl group on the terminal nitrogen, rather than an ethyl group, and a methyl group on the p-amino group of metoclopramide both enhanced the activity. The resulting N-[2-(N-benzyl-N-methylamino)ethyl]-5-chloro-2-methoxy-4-(methylamino) benzamide(23) was about 15 times more active than metoclopramide. In the cyclic series, particularly among the benzamides of 1-benzyl-3-aminopyrrolidine, most of the compounds tested were more active than the corresponding linear benzamides. cis-N-(1-Benzyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4-(methylamino) benzamide (YM-09151-2, 55) was the most active among all of the compounds tested, being 13 and 408 times more potent than haloperidol and metoclopramide, respectively. Moreover, compound 55 exhibited a fairly high ratio of antistereotypic activity to cataleptogenicity compared with haloperidol and metoclopramide. It is expected that compound 55 may be used as a potent drug with few side effects in the treatment of psychosis.