T-bet regulates differentiation of forkhead box protein 3+ regulatory T cells in programmed cell death-1-deficient mice.

Programmed cell death-1 (PD-1) plays an important role in peripheral T cell tolerance, but whether or not it affects the differentiation of helper T cell subsets remains elusive. Here we describe the importance of PD-1 in the control of T helper type 1 (Th1) cell activation and development of forkhead box protein 3 (FoxP3(+)) regulatory T cells (Tr(egs)). PD-1-deficient T cell-specific T-bet transgenic (P/T) mice showed growth retardation, and the majority died within 10 weeks. P/T mice showed T-bet over-expression, increased interferon (IFN)-γ production by CD4(+) T cells and significantly low FoxP3(+) T(reg) cell percentage. P/T mice developed systemic inflammation, which was probably induced by augmented Th1 response and low FoxP3(+) T(reg) count. The study identified a unique, previously undescribed role for PD-1 in Th1 and T(reg) differentiation, with potential implication in the development of Th1 cell-targeted therapy.

Novel low molecular weight spirodiketopiperazine derivatives potently inhibit R5 HIV-1 infection through their antagonistic effects on CCR5.

Novel low molecular weight spirodiketopiperazine derivatives which potently inhibit R5 human immunodeficiency virus type 1 (HIV-1) infection through their antagonistic effects on CCR5 were identified. One such compound E913 (M(r) 484) specifically blocked the binding of macrophage inflammatory protein-1alpha (MIP-1alpha) to CCR5 (IC(50) 0.002 microm) and MIP-1alpha-elicited cellular Ca(2+) mobilization (IC(50) approximately 0.02 microm). E913 potently inhibited the replication of laboratory and primary R5 HIV-1 strains as well as various multidrug-resistant monocyte/macrophage tropic (R5) HIV-1 at IC(50) values of 0.03 to 0.06 microm. E913 was inactive against T cell tropic (X4) HIV-1; however, when combined with a CXCR4 antagonist AMD-3100, E913 potently and synergistically inhibited the replication of dualtropic HIV-1 and a 50:50 mixture of R5 and X4 HIV-1. Antagonism in anti-HIV-1 activity was not seen when E913 was combined with the reverse transcriptase inhibitor zidovudine or protease inhibitors. E913 proved to compete with the binding of antibodies to CCR5 which recognize the C-terminal half of the second extracellular loop (ECL2B) of CCR5. E913 and its analogs are acid-resistant and orally bioavailable in rodents. These data warrant that spirodiketopiperazine derivatives be further developed as potential therapeutics for HIV-1 infection.

Beta-amyloid peptides inhibit acetylcholine release from cholinergic presynaptic nerve endings isolated from an electric ray.

We investigated the effects of beta-amyloid (Abeta) peptides on cholinergic synaptosomes isolated from the electric organ of the Japanese marine ray Narke japonica. Fresh and pre-incubated solutions of Abeta(1-42) inhibited acetylcholine (ACh) release from the synaptosomes evoked by high [K+] depolarization when incubated with synaptosomes for 10 min before the depolarizing stimulus. A freshly prepared solution of Abeta(1-40) did not inhibit the evoked ACh release, but prolonged pre-incubation of Abeta(1-40) solution caused the inhibition. Abeta(1-15) neither in fresh nor pre-incubated solution inhibited. These results have demonstrated that Abeta peptides can acutely inhibit the depolarization-evoked release of ACh by acting directly on cholinergic presynaptic nerve endings. The electrophoresis analysis showed a strong correlation between Abeta aggregation and its inhibition for ACh release.

Pure red cell aplasia after major ABO-incompatible bone marrow transplantation: two case reports of treatment with recombinant human erythropoietin.

A 34-year-old man with acute myelocytic leukemia (AML: MO) and a 32-year-old woman with AML: M2 developed pure red cell aplasia (PRCA) after receiving a major ABO incompatible bone marrow transplant (BMT). The first patient responded to recombinant human erythropoietin (rhEPO) therapy, while the second did not. The second patient also received methylprednisolone (m-PSL) but developed reticulocytosis and hemolysis after the administration of m-PSL. Plasmapheresis was then performed and the patient promptly recovered from hemolysis and PRCA. We conclude that close attention must be paid when treating PRCA following major ABO-incompatible BMT with rhEPO and m-PSL, as there is always the potential for massive hemolysis.

Rationally designed syntheses of high-mannose and complex type undecasaccharides.

Synthetic routes are described to a high-mannose type triantenary undecasaccharide 1 and a completely protected form 39 of the complex type biantenary undecasaccharide 2 carrying alpha-(2-->3)-linked sialic acid residues. Starting from a previously reported trisaccharide 4, the core pentasaccharides 15 and 37 were synthesized through regio- and/or stereo-selective mannosylation with a suitably protected mannosyl donor. Chain elongation of 15 by stepwise addition of a mannose residue afforded an undecasaccharide 20 that was eventually deprotected to give 1. On the other hand, coupling of 38 and a trisaccharide glycosyl donor 36 afforded 39.

Correlation between myocardial dysfunction and changes in myosin isoenzymes in diabetic rat hearts.

We investigated the relationship between papillary muscle function and the myosin isoenzyme pattern, collagen content, and the type of myocardial collagen in diabetic rats to elucidate the mechanism of short-term myocardial dysfunction in diabetes. Diabetes was induced in 9-week-old male Wistar rats with a single intravenous injection of streptozotocin. One-half of the diabetic rats were treated with insulin. Age-matched control rats were also studied. The time to peak tension (TPT) of isometric papillary muscle contraction, time to 1/2 relaxation, and time from the peak tension to the peak decrease in tension (TPN) were significantly prolonged in diabetic rats at 4, 8, and 12 weeks. The peak increase and decrease in tension were slower in the diabetic rats compared with control rats. The level of the myocardial myosin isoenzyme V3 was greater in diabetic rats than in control rats at each interval. Findings in insulin-treated rats were similar to those in controls. The collagen content and the ratio of type I collagen to type III collagen were similar in all groups. The V3 level was significantly correlated with mechanical parameters (TPT versus %V3: r = 0.81, p < 0.01; TPN versus % V3: r = 0.78, p < 0.01). Our findings suggest that short-term myocardial dysfunction in diabetic rats is related to changes in the myosin isoenzyme pattern.

Hyperthyroidism complicated with sick sinus syndrome.

A 50-year-old woman was transported to a hospital complaining of marked general malaise and epigastralgia with diarrhea and vomiting. Her electrocardiogram showed sinus arrest with a duration of nearly 8 sec. Atrial pacing with an external pacemaker improved her symptoms promptly. Following transfer to our hospital 3 days later, bradyarrhythmia was not detected despite the removal of the external pacemaker. Transient atrial fibrillation was found in our hospital, and she was diagnosed as hyperthyroidism based on findings of finger tremor, exophthalmos, diffuse goiter and an abnormally high level of thyroid hormone. On cardiac catheterization, left ventriculography showed anterior wall hypokinesis and mild mitral regurgitation. Coronary arteriography showed the absence of organic stenosis. Right ventricular endomyocardial biopsy showed myocardial hypertrophy and partial disarray, but no findings of myocarditis. Electrophysiological study showed the normal upper range of AH-time (120 msec) and HV-time (50 msec), and prolongation of corrected sinus recovery time (CSRT, 955 msec). After a euthyroid state was successively induced for about 10 days by methylmercaptoimidazole therapy, AH-time, HV-time and CSRT were shortened to 85, 35 and 290 msec respectively. Her complaints and sick sinus syndrome disappeared after the treatment of hyperthyroidism without a pacemaker.

Genomic organization of mouse J kappa recombination signal binding protein (RBP-J kappa) gene.

We have isolated a cDNA clone (RBP-2) for the protein (RBP-J kappa) which binds to immunoglobulin recombination signals with 23-base pair spacers (Matsunami, N., Hamaguchi, Y., Yamamoto, Y., Kuze, K., Kangawa, K., Matsuo, H., Kawaichi, M., and Honjo, T. (1989) Nature 342, 934-937). During further screening of a cDNA library from the same mouse pre-B cell line (38B9), we have isolated a second cDNA clone (RBP-2N) which differs from RBP-2 in its 5' sequence. RNase protection assays indicated that the RBP-2N type mRNA was produced in 10-20 times the quantity as RBP-2 mRNA. To elucidate the relationship between these two mRNAs, we analyzed the genomic organization of the RBP-J kappa gene. Southern hybridization of mouse genomic DNA detected at least 7 EcoRI fragments hybridizing to an RBP-2 cDNA probe, suggesting a complex structure for the RBP-J kappa gene. Cloning of each EcoRI fragment revealed one functional RBP-J kappa gene and three related genes. The functional gene was composed of 11 exons and spanned at least 50 kilobase pairs. The sequence of exon 1 and its 5'-flanking region contained a GC-rich promoter-like region but no apparent TATA box. The initiation site of transcription was heterogeneous, and the two types of mRNA are produced from the same exon by transcription initiation at different sites and by different usage of splice signals. Two of the three related genes were processed pseudogenes with scattered stop codons. The other was also a processed gene with a sequence exactly the same as that of RBP-2, except that this gene lacked the sequence corresponding to the first exon of the functional gene.

Stereoselective total synthesis of glycopeptides bearing the dimeric and trimeric sialosyl-Tn epitope.

The dimeric and trimeric sialosyl-Tn epitopes, [alpha-D-Neup5Ac-(2----6)-alpha-D-GalpNAc-(1----3)-L-Ser]n-L-Val (n = 2 and 3), which represent part of a clustered carbohydrate region of glycophorin A, a human erythrocyte glycoprotein, have been synthesised stereoselectively. 2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-D-galactopyranosyl fluoride (GalpNAc unit), Fmoc-L-serine phenacyl ester (Ser unit), and benzyl 5-acetamido-4,7,8,9-tetra-O-benzyl-5-deoxy-3-S-phenyl-3-thio-D-erythro-L - gluco-2-nonulopyranosylonate bromide (Neup5Ac unit) were the key intermediates for stereoselective glycosylation. 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline-promoted elongation of the peptide chain and then hydrogenolysis afforded the title compounds.

Increase in left ventricular chamber stiffness in patients with non-insulin dependent diabetes mellitus.

Indices of left ventricular ejection and diastolic filling were measured by cineventriculography in 11 patients with non-insulin dependent diabetes mellitus without significant coronary stenosis and 11 control subjects without diabetes mellitus. Indices of left ventricular ejection, such as ejection fraction and peak ejection rate, were the same in the two groups. The left ventricular end-diastolic volume index and the rapid filling volume index were significantly smaller, the peak filling rate was lower, the left ventricular end-diastolic pressure was higher and the modulus of left ventricular chamber stiffness was larger in the diabetic patients than in the control subjects. These results indicate that left ventricular chamber stiffness is increased in patients with non-insulin dependent diabetes mellitus.

Correlation between histopathological changes and mechanical dysfunction in diabetic rat hearts.

Recent clinical and experimental studies have suggested that diabetic patients may develop myocardial dysfunction in the absence of coronary heart disease and hypertension. In this study, the correlation between histopathological changes and myocardial dysfunction was studied in experimental diabetic rat hearts. Male Wistar rats were made diabetic at 9 weeks of age with a single intravenous injection of streptozotocin 50 mg/kg. The diabetic rats were studied along with age-matched control and insulin-treated rats at 4, 8, 12 and 24 weeks after the induction of diabetes to investigate isolated papillary muscle contraction and the histopathological picture simultaneously. In the isometric contractions, resting and developed tensions were similar. Time to peak tension and time to 1/2 relaxation were prolonged and the peak rate of tension rise and tension fall was depressed. On histological examination of left ventricular walls, diameters of myocytes were similar at all disease durations. Interstitial fibrosis and disarrangement of myocytes after 12 weeks were slightly increased in the diabetic hearts. Mechanical parameters did not worsen in parallel with the duration of diabetes and histological changes, but correlated with the blood glucose level. These data suggest that short-term mechanical defects in the experimental diabetic rat heart result from the metabolic disorder itself, with histopathological changes occurring later.

An autopsy case of hypertrophic cardiomyopathy showing dilated cardiomyopathy-like features by serial ventriculography.

An autopsy case of hypertrophic obstructive cardiomyopathy (HOCM), in which the clinical features of dilated cardiomyopathy (DCM) developed after 10 years is described. Three serial left ventriculograms (LVG) and bi-ventriculograms (BVG) were performed during the 10-year follow-up. At the time of the first observation, the ECG revealed a giant negative T wave (GNT) in V5 (-1.5mV) and high voltage (SV1 + RV5 = 8.1mV). The LVG and BVG showed concentric left ventricular wall hypertrophy and a pressure gradient in the left ventricular out-flow tract. However, follow-up studies disclosed resolution of the GNT with decreased high voltage. All wall thicknesses and the ejection fraction were significantly decreased and the wall motion was reduced serially. These findings were consistent with the clinical profile of DCM. At autopsy, the heart weighed 610 g and showed dilated change of the left ventricular cavity. Significant myocardial fibrosis, bizarre myocardial hypertrophy with disorganization (BMHD) and severe narrowing of the intramural small arteries due to intimal thickening were found in the left ventricular wall, especially in the anterior and interventricular septal wall. This autopsy case suggests that some cases of HCM, with serial disappearance of the deep negative T wave and high voltage, deteriorate to the clinical profile of DCM.

[Early diastolic dysfunction of the left ventricle affected by hypertrophy and abnormal histopathology in hypertrophic cardiomyopathy].

In this study, we investigated correlations of left ventricular hypertrophy and its histopathology with diastolic dysfunction in patients with hypertrophic cardiomyopathy. Nine control subjects and 14 hypertrophic cardiomyopathy (HCM) patients with asymmetrical septal hypertrophy were evaluated. M-mode echocardiography was used to assess fractional shortening (FS), isovolumic relaxation time (IRT), and the left ventricular filling volume index during rapid and slow filling periods and atrial contraction period (RFVI, SFVI and ACVI). End-diastolic thickness of the interventricular septum and posterior wall was determined using biventriculography. Right ventricular endomyocardial biopsies were performed to calculate the diameters of myocytes, the percentage of fibrosis and the eccentricity e which indicates the degree of myocardial disarrangement including disorganization. The FS was normal in the two groups. The IRT of the HCM group was significantly greater and the RFVI significantly less than those of the controls. The left ventricular wall thickness, the diameters of myocytes and the percentage of fibrosis in the HCM group were significantly greater; and the eccentricity e was significantly less, suggesting that myocardial disarrangement was significantly more severe than that in the controls. Significant positive correlations were observed between the IRT and the wall thickness (r = 0.647), between the diameter of myocytes (r = 0.681) and the percentage of fibrosis (r = 0.628), and there was a significantly negative correlation between the IRT and the eccentricity e (r = -0.759). There was a significantly negative correlation between the RFVI and the wall thickness (r = -0.663); and a significantly positive correlation between the RFVI and the eccentricity e (r = 0.579). Multiple regression analyses showed that the diameter of myocytes, the percentage of fibrosis and the eccentricity e all correlated significantly with the IRT (R = 0.821) and the RFVI (R =0.604). The standard regression coefficients of the diameter of myocytes, the percentage of fibrosis and the eccentricity e were 0.253, 0.278 and -0.431 in respect to IRT, and those of the percentage of fibrosis and the eccentricity e were -0.204 and 0.469 in respect to RFVI, respectively. These results indicated that diastolic dysfunction in hypertrophic cardiomyopathy is related not only to the degree of left ventricular hypertrophy, but also to the degree of myocardial hypertrophy, increased interstitial fibrosis, and especially to myocardial disarrangement including disorganization.

[A noninvasive study on characteristics of the left ventricular function in elderly hypertensive patients].

To characterize the left ventricular (LV) systolic and diastolic function in elderly hypertensives, mechano-and echocardiography was performed in 27 elderly patients more than 65 years of age (20 hypertensives and 7 normotensives) and 46 middle-aged patients (34 hypertensives and 12 normotensives). In normotensives, a positive correlation was found to exist between age and blood pressure, LV wall thickness, II-RF interval and the A/E ratio, whereas the correlation of age to systolic parameters such as ET/PEP and EF was absent. This suggests that the LV diastolic function was impaired progressively by aging with preservation of the LV systolic function. In elderly hypertensives, LV systolic function was deteriorated and the A/E ratio increased as diastolic blood pressure became elevated. The A/E ratio also increased in this group as compared with that in middle-aged hypertensives, although the differences was not statistically significant. Unlike those in middle-aged hypertensives, the indices of LV function except the elevated blood pressure and the increased cardio-thoracic ratio were not significantly different between hypertensive and normotensive elderly patients. This might be because of the wide range of normal values in elderly persons due to the various degrees of atherosclerotic changes and other pathophysiologic factors, which might have masked obvious characteristics of hypertension in elderly hypertensives.

Thyroid antigen-mediated glomerulonephritis in Graves' disease.

We describe a patient with Graves' disease in whom marked proteinuria, microhematuria and hypoalbuminemia were associated. Renal biopsy demonstrated electrondense deposits in the capillary basement membrane, a finding consistent with immune complex glomerulonephritis. Indirect immunofluorescent examination with rabbit antihuman thyroglobulin indicated that these electron-dense deposits were thyroid antigen-mediated immune complexes.

Structure of the horseradish peroxidase isozyme C genes.

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.

A total synthesis of hematoside, alpha-NeuGc-(2----3)-beta-Gal-(1----4)-beta-Glc-(1----1)-Cer.

Methyl (5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5- dideoxy-D-glycero-beta-D-galacto-2-nonulopyranosyl chloride)onate, prepared from N-glycolyl-neuraminic acid, was used for the glycosylation of benzyl O-(2,6-di-O-benzyl-beta-D- galactopyranosyl)-(1----4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside to give benzyl O-[methyl (5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- alpha- and beta-D-galacto-2-nonulopyranosyl)onate]-(2----3)-O-(2,6-di-O -benzyl-beta- D-galacto-pyranosyl)-(1----4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside, and a regioisomer. Compounds 9 and 13 were converted into the corresponding glycotriaosyl glycosyl donors which, upon coupling with (2S,3R,4E)-3-O-tert-butyldiphenylsilyl-2-N- tetracosanoylsphingenine, afforded completely protected hematoside and a stereoisomer 26, respectively. Deprotection of 22 and 26 completed the first total synthesis of both hematoside and a stereoisomer, beta-NeuGc-(2----3)-beta-Gal-(1----4)-beta-Glc-(1----1)-Cer.

Synthesis of diastereomeric mixture of 15,19,23-trimethylheptatriacontane, contact sex pheromone of tsetse fly,Glossina morsitans morsitans westwood.

A nine-step synthesis is described for a diastereomeric mixture of 15,19,23-trimethylheptatriacontane, a contact sex pheromone ofGlossina morsitans morsitans Westwood. The compound has been synthesized by means of double alkylation of diethyl 3-oxoglutarate (DEOG) with 3-methyl-2-heptadecenyl methanesulfonate, which was readily prepared from 1-hexadecene, as the key step.