Ezetimibe improves postprandial hyperlipidaemia in patients with type IIb hyperlipidaemia.

BACKGROUND: Postprandial hyperlipidaemia is known to be a high-risk factor for atherosclerotic disease because of rapid and lasting accumulations of triglyceride-rich lipoproteins and remnants. The Niemann-Pick C1-Like 1 (NPC1L1) protein acts as an intestinal cholesterol transporter and ezetimibe, which inhibits NPC1L1, has been used in patients with hypercholesterolaemia. We investigated effects of ezetimibe on fasting lipid and lipoprotein profiles and postprandial hyperlipidaemia in patients with type IIb hyperlipidaemia. MATERIALS AND METHODS: Ezetimibe 10 mg per day was administered in ten patients with type IIb hyperlipidaemia for 2 months, and lipid and lipoprotein profiles were examined during fasting and after an oral fat loading (OFL) test. RESULTS: In the fasting state, ezetimibe significantly decreased not only total cholesterol, low density lipoprotein (LDL)-cholesterol and apolipoproteinB-100 (apoB-100) levels but triglycerides (TG), apoB-48 and remnant lipoprotein cholesterol (RemL-C) levels. High performance liquid chromatography analysis showed that ezetimibe decreased cholesterol and TG levels in the very low density lipoprotein (VLDL) and LDL size ranges as well as apoB-100 levels, suggesting a decrease in numbers of VLDL and LDL particles. After OFL, ezetimibe decreased the area under the curve for TG, apoB-48 and RemL-C. Ezetimibe decreased postprandial elevations of cholesterol and TG levels in the chylomicrons (CM) size range, suggesting that the postprandial production of CM particles was suppressed by ezetimibe. CONCLUSIONS: These findings suggest that ezetimibe improves fasting lipoprotein profiles and postprandial hyperlipidaemia by suppressing intestinal CM production in patients with type IIb hyperlipidaemia and such treatment may prove to be effective in reducing atherosclerosis.

Cryptococcal pleuritis developing in a patient on regular hemodialysis.

A 64-year-old male on regular hemodialysis who was a human T lymphotrophic virus Type I (HTLV-I) carrier developed cryptococcal pleuritis. The initial manifestations of the present case were a persistent cough and the accumulation of unilateral pleural effusion. A culture of the pleural fluid of the patient grew cryptococcus neoformans and a test for antigens against cryptococcus neoformans in the pleural fluid was also positive, therefore, cryptococcal pleuritis was diagnosed. Pleural cryptococcosis per se is rare and it is extremely rare for a dialysis patient to develop pleural cryptococcosis. To our knowledge, only a few cases of cryptococcal pleuritis have so far been reported in patients on dialysis. Furthermore, an isolated occurrence of cryptococcal pleuritis with no cryptococcal pulmonary parenchymal lesions, as was seen in the present case, is rare because cryptococcal pleuritis is usually associated with underlying cryptococcal pulmonary parenchymal lesions. Patients on chronic dialysis are susceptible to developing pleural effusion from many etiologies such as congestive heart failure, infection (tuberculosis, bacterial, viral, parasitic, fungal), collagen vascular disease, drug reaction, metastasis, or uremia itself. Cryptococcal pleuritis developing in a dialysis patient is extremely rare, but physicians should consider cryptococcal infection as a possible cause when pleural effusion develops in a dialysis patient and no other cause is identified, as occurred in the present case.

Vascular endothelial dysfunction resulting from L-arginine deficiency in a patient with lysinuric protein intolerance.

Although L-arginine is the only substrate for nitric oxide (NO) production, no studies have yet been reported on the effect of an L-arginine deficiency on vascular function in humans. Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of dibasic amino acid transport caused by mutations in the SLC7A7 gene, resulting in an L-arginine deficiency. Vascular endothelial function was examined in an LPI patient who was shown to be a compound heterozygote for two mutations in the gene (5.3-kbp Alu-mediated deletion, IVS3+1G-->A). The lumen diameter of the brachial artery was measured in this patient and in healthy controls at rest, during reactive hyperemia (endothelium-dependent vasodilation [EDV]), and after sublingual nitroglycerin administration (endothelium-independent vasodilation [EIV]) using ultrasonography. Both EDV and NO(x) concentrations were markedly reduced in the patient compared with those for the controls. They became normal after an L-arginine infusion. EIV was not significantly different between the patient and controls. Positron emission tomography of the heart and a treadmill test revealed ischemic changes in the patient, which were improved by the L-arginine infusion. Thus, in the LPI patient, L-arginine deficiency caused vascular endothelial dysfunction via a decrease in NO production.

Involvement of CPI-17 downregulation in the dysmotility of the colon from dextran sodium sulphate-induced experimental colitis in a mouse model.

The mechanism of gastrointestinal dysmotility in inflammatory bowel disease has not been clarified. In this study, we examined the mechanism involved in the inflamed distal colon isolated from a mouse model of dextran sodium sulphate-induced ulcerative colitis (DSS-treated mouse). Although substance P-induced contraction was not changed, carbachol-induced contraction was reduced in the DSS-treated mouse colon. Pre-incubation with the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) or the cyclooxygenase inhibitor indomethacin did not reverse the carbachol-induced contraction in the DSS-treated mouse colon. In semi-quantitative reverse transcription-polymerase chain reaction experiments and Western blot analysis, muscarinic M3 receptor expressions were not changed. The Ca2+ -sensitization of contractile elements induced by carbachol with GTP or GTPgammaS was reduced in the beta-escin-permeabilized DSS-treated mouse colon. Although the expression of proteins such as rhoA, ROCK1, ROCK2 or MYPT1 in smooth muscles was not changed, the expression of CPI-17, the functional protein involved in smooth muscle Ca2+ -sensitization, was significantly decreased in the DSS-treated mouse colon. These results suggest that the suppression of carbachol-induced contraction in mice with colitis is attributable at least partially to the increased activity of myosin phosphatase following the downregulation of CPI-17.

Role of TNF-alpha in muscularis inflammation and motility disorder in a TNBS-induced colitis model: clues from TNF-alpha-deficient mice.

Macroscopic and histological analysis revealed that the colonic inflammation induced by 2,4,6-trinitrobenzenesulphonic acid (TNBS) was of lower grade in tumour necrosis factor-alpha (TNF-alpha)(-/-) mice than in wild-type mice. Myeloperoxidase activity, an indicator of neutrophilic infiltration, was also low in both the mucosal and smooth muscle layer of the TNF-alpha(-/-) mouse colon. After the induction of inflammation with TNBS, the levels of proinflammatory cytokines, such as TNF-alpha, interleukin-1beta and interleukin-6, were elevated both in the inflamed mucosa and muscle layers in the wild-type mice; however, the productions of these cytokines were greatly reduced in the TNF-alpha(-/-) mouse colon. The contractions of isolated colonic smooth muscle strips induced by several stimulatory agents were significantly decreased after treatment with TNBS in wild-type mice; however, these contractions were scarcely affected in TNF-alpha(-/-) mice. Finally, using the organ culture method, we found that TNF-alpha directly (independent of mucosal inflammation) disturbs the smooth muscle function. These results suggest that TNF-alpha plays an essential role not only in mucosal inflammation but also in muscularis inflammation in the colon of mice with TNBS-induced colitis, and that TNF-alpha directly induces motor dysfunctions by acting on the smooth muscle.

Unassigned or nonsense codons in Micrococcus luteus.

We previously reported that in Micrococcus luteus, a Gram-positive eubacterium with high genomic G + C content, certain codons ending with A did not appear in coding frames, including termination sites, and tRNAs that translate these codons were not detected. These facts suggest that at least some of them are unassigned (nonsense) codons, i.e. not assigned to any amino acid or to any stop signal. We have investigated whether AGA and AUA, universal Arg and Ile codons, respectively, are really unassigned codons by using a cell-free extract prepared from M. luteus and synthetic messenger RNAs. Translation of synthetic mRNA containing in-frame AGA codons does not result in "read-through" to codons beyond the AGA codons, i.e. translation ceases at codon AGA. Essentially the same result was obtained with mRNA containing AUA in-frame. A sucrose-gradient centrifugation profile of the reaction mixture has shown that practically all of the peptides that have been synthesized are attached to 70 S ribosomes. When in-frame AGA or AUA codons are replaced by UGA codons in mRNA, no read-through occurs beyond UGA, just as in the case of AGA or AUA. However, the synthesized peptide is released from the 70 S ribosomes. These data suggest that AGA and AUA are unassigned codons and differ from UGA in that they are not used for termination.

Novel anticodon composition of transfer RNAs in Micrococcus luteus, a bacterium with a high genomic G + C content. Correlation with codon usage.

The number and relative amount of isoacceptor tRNAs for each amino acid in Micrococcus luteus, a Gram-positive bacterium with high genomic G + C content, have been determined by sequencing their anticodon loop and its adjacent regions and by selective labelling of tRNAs. Thirty-one tRNA species with 29 different anticodon sequences have been detected. All the tRNAs have G or C at the anticodon first position except for tRNA(ICGArg) and tRNA(NGASer), in response to the abundant usage of NNC and NNG codons. No tRNA with the anticodon UNN capable of translating codon NNA has been detected, in accordance with a very low or zero usage of NNA codons. The relative amount of isoacceptor tRNAs for an amino acid determined by selective labelling strongly correlates with usage of the corresponding codons. On the basis of these and other observations in this and other eubacterial species, we conclude that the relative amount and anticodon composition of isoacceptor tRNA species are flexible, and their changes are mainly adaptive phenomena that have been primarily affected by codon usage, which in turn is affected by directional mutation pressure.

Translation of synonymous codons in family boxes by Mycoplasma capricolum tRNAs with unmodified uridine or adenosine at the first anticodon position.

In Myocoplasma capricolum, codon family boxes except for arginine and threonine (CUN leucine, GUN valine, UCN serine, CCN proline, GCN alanine, and GGN glycine) have only a single tRNA species with the anticodon sequence UNN (N is U, C, A or G), the first nucleoside U being unmodified. Incorporation of the [3H]amino acid into the peptide fraction was examined with the M. capricolum cell-free translation system. Synthetic mRNA containing each of the respective amino acid codons in the coding frame was subjected to translation. The tRNAUNN translated all the family box codons with similar, if not equal, efficiencies. In M. capricolum, there are two species of threonine tRNA, tRNA(UGUThr) and tRNA(AGUThr), the first nucleoside U or A being unmodified. The tRNA(UGUThr) species translates codons ACA, ACG and ACU efficiently, and ACC only poorly. In contrast, the tRNA(AGUThr) species translates codons ACU, ACC and ACG efficiently and ACA poorly.

Distribution of cognates of group II introns detected in mitochondrial cox1 genes of a diatom and a haptophyte.

We identified group IIA introns that contain an open reading frame (ORF) in the mitochondrial cytochrome oxidase subunit I (cox1) genes of yellow algae, a diatom Thalassiosira (Th.) nordenskioeldii CCMP 992 collected from the east coast of USA, and a haptophyte Pavlova (Pa.) lutheri CCMP 1325 collected from Finland. Cognate introns of CCMP 1325 were detected in all Pa. lutheri strains investigated, which were collected from various oceans. In contrast, the intron was absent from closely related species belonging to the same genus Pavlova. This was also the case for the group II intron detected in a diatom Th. nordenskioeldii CCMP 992. The group II intron of CCMP 992 was located at the corresponding site to the group IIA intron found in Pylaiella (synonym, Pilayella) littoralis. The deduced secondary structures of these introns, one of which is from a diatom and the other from a brown alga, were virtually identical. In contrast, the haptophyte group II intron was inserted at a novel locus, and shares no particularly high sequence homology with any intron known to date. The phylogenetic tree based on the intronic ORF domain was not congruent with that based on the cox1 exon. The most prominent property of the intronic ORF tree was that introns located at homologous sites made robust pair clades irrespective of the phylogenetic relationships of the organisms. This suggests that mitochondrial group II introns often invade intronless alleles across the species barrier with site specificity. Homology analysis of the haptophyte intronic ORF suggested that it comprises three domains: reverse transcriptase (RT), RNA maturase (Ma), and H-N-H endonuclease. However, the intronic ORF of the diatom contains the Ma domain but is apparently missing the H-N-H domain, and its RT domain is most probably partly or completely lacking in function.

Distinctive origins of group I introns found in the COXI genes of three gree algae.

Upon surveying the cytochrome c oxidase subunit I (COXI) gene of green algae, we found group I introns in three species of algae, Chlorella vulgaris (Cv), Scenedesmus quadricauda (Sq) and Protosiphon botryoides (Pb). The comparative analysis of these nucleotide sequences and their secondary structures revealed that the introns of Cv, Sq, and Pb belong to groups IB1, ID, and IB2, respectively. Each of the three introns contained an open reading frame (ORF) that showed a similarity to the sequence of the LAGLIDADG endonuclease family. However, each of the intronic ORFs in Sq and Pb had a discontinuity in the middle of' the sequences coding for the LAGLIDADG endonuclease. Either of the two ORFs could be restored to a sequence homologous to the LAGLIDADG endonuclease by the insertion of a nucleotide in the appropriate position. In Sq, a putative pseudo-knot structure was detected in the intronic ORF This suggests the occurrence of a ribosomal frameshift in the translation of the ORF. because such pseudo-knot structures are common in viral ORFs employing a (-1) ribosomal frameshift. In the phylogenetic tree that was inferred from the amino acid sequences of algal and non-algal intronic ORFs, the three algal ORFs did not make a cluster, but were scattered throughout the tree. In addition. each of the three algal ORFs showed a close relationship to the ORFs of non-algal introns that were inserted at the corresponding site of the COX] gene, suggesting distinctive origins of the three algal introns via independent horizontal transfers.

Evolutionary changes in the genetic code.

The genetic code has been influenced by directional mutation pressure affecting the base composition of DNA, sometimes in the direction of increased GC content and at other times, in the direction of AT. Such pressure led to changes in species-specific usages of codons and tRNA anticodons, and also in amino acid assignments of codons in mitochondria and in several intact organisms. These code changes are probably recent evolutionary events. The genetic code is not 'frozen', but instead it is still evolving.

Deficiency of apolipoprotein B synthesis in Suncus murinus.

We reported previously that fatty liver is easily induced in a novel experimental animal, Suncus murinus (suncus) by withholding food. In this study, we focused on lipoprotein and apolipoprotein secretion from the liver. The study of lipoproteins from this animal revealed that small amounts of lipoproteins with apolipoprotein (apo) E but without apo B were observed in the fraction of density less than 1.08 g/ml. In order to learn whether apo B is synthesized by the liver or not, isolated suncus livers were perfused with an addition of [35S]methionine. Small amounts of radioactivity were observed in apo E of VLDL, and fairly large amounts in apo E and A-I in the fraction of LDL + HDL, suggesting that VLDL was secreted with apo E but not with apo B from the liver. Northern blot analysis with use of rat apo B cDNA revealed a weak signal of hybridized rat apo B cDNA between 15 kb and 9 kb in the suncus liver and intestinal mucosa; this is almost the same size as rat apo B mRNA. This finding suggests the presence of apo B mRNA in the suncus. In conclusion, apo B is not secreted from the suncus liver, owing to a defect in intracellular post-transcriptional processing or to ineffective transcription. This might be one of the reasons for fatty deposits in the suncus liver. Suncus may be a candidate for an animal model of abetalipoproteinemia as well as fatty liver due to a defect of apo B synthesis.

Effect of starving and refeeding on lipid metabolism in suncus.

We have previously reported that fatty liver is easily induced in a novel experimental animal, Suncus murinus (suncus) by withholding food, and that apolipoprotein B (apo B) is not actively synthesized in the liver. In the present paper we describe the effect of starving and refeeding on lipid and lipoprotein metabolism in suncus, in order to explore the mechanisms of induction of fatty liver by starving and of its improvement by refeeding. Starvation induced increase in triglyceride content and decrease in glycogen content of the liver. Although the glycogen content returned to the level before starvation at 12 h after refeeding, the triglyceride content decreased gradually but did not reach the prestarvation level even at 24 h after refeeding in suncus. Plasma lipids, glucose, and insulin levels were decreased by starvation and returned to the levels before starvation between 8 and 24 h after refeeding. On the other hand, the plasma levels of free fatty acid and ketone bodies were elevated significantly by starvation and decreased rapidly by refeeding. These responses to starvation and refeeding, except for the change in hepatic triglyceride, are in common with other experimental animals, suggesting that there are no abnormalities in glucose metabolism or in fatty acid metabolism in suncus. In conclusion, the fatty liver induced by starvation in suncus may be caused by impaired triglyceride transport out of the liver, for which apolipoprotein B is mostly responsible, as reported previously.

Characterization of serum lipoproteins from Suncus: a candidate animal model for abetalipoproteinemia.

We have previously reported that fatty liver was induced in a novel experimental animal, Suncus murinus (suncus), by 24-h fasting and that apolipoprotein B (apo B) was not actively synthesized in the liver. However, a faint signal of apo B mRNA was detected in the liver, suggesting possible synthesis of apo B. Small amounts of VLDL and LDL have been separated from suncus serum by ultracentrifugation. Electron microscopic study of the lipoproteins revealed the existence of small particles in VLDL. High performance liquid chromatographic analysis of the lipoproteins showed that the peaks of TG and cholesterol were mainly at the HDL fraction. These results indicate the existence of lipoproteins as small as HDL which were rich in TG and floated at the density of VLDL upon ultracentrifugation. Apolipoprotein analysis showed two bands of 500- and 200-kDa proteins in VLDL and LDL. Western blot analysis using antibody against the 500-kDa protein revealed reaction not only with suncus 500- and 200-kDa proteins but also with human apo B-100. In conclusion, a small amount of apo B is transported in the suncus serum as VLDL and LDL, although almost all lipid is packed in HDL-size particles.

[Congenital tracheal stenosis due to complete cartilage rings with right pulmonary agenesis].

A 2-month-old male infant with severe dyspnea was diagnosed as having right pulmonary agenesis at birth and was admitted to our hospital after tracheal intubation with an endotracheal tube of 3 mm in diameter. However, the trachea was too stenotic to place the tube in the proper position. Chest X-ray on admission showed pneumonia of the left lung. Preoperative chest computed tomography (CT) scan and bronchoscopy showed that from the level of 12 mm beneath the coricoid cartilage, the trachea tapered and continuing to the tracheal carina and that the smallest tracheal level was located 18 mm distal from the coricoid cartilage, the area of which was 4 mm2. His respiratory condition rapidly deteriorated in spite of intravenous administration of antibiotics and mechanical ventilation. Percutaneous cardiopulmonary support (PCPS) was used to maintain his pulmonary function, and pericardial tracheoplasty was performed. Chest X-ray immediately after the operation did not show left lung reexpansion due to severe pulmonary edema. High-dose steroid pulse therapy was performed, but it was not effective. He died from acute respiratory failure due to infantile respiratory distress syndrome (IRDS) on postoperative day 3. The outcome in this case shows that it is very risky to repair tracheal stenosis in a patient with pneumonia using PCPS.

Synchronous phosphorylation of CPI-17 and MYPT1 is essential for inducing Ca(2+) sensitization in intestinal smooth muscle.

BACKGROUND: Myosin phosphatase activity is regulated by mechanisms involving the phosphorylation of CPI-17 and MYPT1, primarily based on studies with tonic-type vascular smooth muscles. This study examined how these mechanisms contribute to the regulation of contraction of a phasic-type intestinal smooth muscle. METHODS: Phosphorylation levels, tension, and Ca(2+) sensitization was detected in rat ileal smooth muscle. Key Results In rat ileal smooth muscle, phosphorylation level of CPI-17 at Thr(38) and MYPT1 at Thr(853) , but not MYPT1 at Thr(696) , were increased with carbachol (1µmolL(-1) ) accompanied with muscle contraction. The PKC inhibitor Go6976 (1µmol L(-1) ) inhibited the carbachol-induced phosphorylation of CPI-17, whereas the Rho-associated kinase (ROCK) inhibitor, Y-27632 (10µmol L(-1) ) inhibited the carbachol-induced phosphorylation of both CPI-17 and MYPT1. Application of Go6976 or Y-27632 alone inhibited the carbachol-induced contraction; however, the combined application of these inhibitors did not inhibit the contraction in an additive manner. In ß-escin-permeabilized ileal strip, treatment with antiphosphorylated antibodies for CPI-17 at Thr(38) and MYPT1 at Thr(853) and Thr(696) alone almost completely abolished the Ca(2+) sensitization due to carbachol with GTP. CONCLUSIONS & INFERENCES: In conclusion, receptor stimulation increases the Ca(2+) sensitivity of contractile elements through CPI-17 phosphorylation via the PKC/ROCK pathways and MYPT1 phosphorylation via the ROCK pathway, when these mechanisms operate cooperatively and/or synchronously in intestinal smooth muscle.