The contribution of the asymmetric alpha 1beta 1 half-oxygenated intermediate to human hemoglobin cooperativity.

Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O(2)] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O(2))beta(Fe-O(2))][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O(2) binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O(2) equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O(2)-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)]( 2) and [alpha(Zn)beta(Fe)]( 2) were measured by using an Imai apparatus. It was shown that the first and second O(2) equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg(-1), respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O(2) binding study.     

A simple and efficient synthesis of puromycin, 2,2′-anhydro-pyrimidine nucleosides,cytidines and 2′,3′-anhydroadenosine from 3′,5′-O-sulfinyl Xylo-nucleosides

Synthesis of antibiotics, puromycin and 3 ′-amino-3 ′-deoxy-N ⁶,N ⁶-dimethyladenosine 11 was achieved by utilizing the cyclic sulfite 6a of the xylo-3 ′,5 ′-dihydroxy group as a new protective group. The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization of 2-α-carbamate 7. In a similar manner, 2,2 ′-anhydro-pyrimidine nucleosides 15, ribo-cytidines 17 and 2 ′,3 ′-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4, 5, and 6b, respectively.     

Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

Effects of external calcium, magnesium, and temperature on spontaneous contractions of pregnant human myometrium

Spontaneous isometric contractions of small isolated segments from the isthmic region of pregnant human myometrium were recorded to clarify the characteristics of and influences of experimental conditions on contractions. There is a spontaneous periodicity in membrane activity of human myometrium, and contractions evoked with a sustained electric stimulus were affected by spontaneous rhythmic contractions. The frequency of contractions increased markedly, but their duration decreased when temperature of the bathing fluid was increased from 26 degrees C to 39 degrees C. Both excess (7 mM) and low (0.5 mM) calcium levels suppressed the generation of spontaneous contraction. Frequency and half-duration gradually decreased when external magnesium was increased from 0 mM to 3.6 mM. These results indicate that the pattern of contraction depends on the frequency of and intervals between each action potential, and that spontaneous contractility-particularly its frequency-is extremely sensitive to external temperature and ionic conditions.     

A new species of Trichogramma (Hymenoptera: Trichogrammatidae) parasitic on eggs of the alderfly Sialis melania (Neuroptera: Sialidae) from Japan,with comments on its phylogeny and male wing polymorphism

A new species of Trichogramma that parasitizes Sialis melania eggs is described as Trichogramma tajimaense Yashiro, Hirose and Honda, sp. nov. from Japan. Its phylogenetic position is based on a DNA‐based analysis, and data regarding its male wing polymorphism are also presented. The view that T. tajimaense is closely related to T. semblidis, another parasitoid of Sialis eggs, is supported by the results of a phylogenetic analysis, as well as by the biological and morphological similarities between both species. Trichogramma tajimaense is also similar in male wing polymorphism to T. kurosuae, a gregarious egg parasitoid of the lepidopteran Ivela auripes, as both Trichogramma species exhibit male wing trimorphism (fully alate, brachypterous and apterous forms) in contrast to the male wing dimorphism (fully alate and apterous forms) of T. semblidis. However, no phylogenetic analysis reveals a close relationship between T. tajimaense and T. kurosuae, and a difference exists between these two species in the mean percentage of flightless (brachypterous and apterous) males that emerge from a host egg mass; 96% of T. tajimaense males are incapable of flight, whereas about 50% of T. kurosuae males are flightless. Because all or almost all males of T. semblidis parasitizing Sialis eggs are apterous, T. tajimaense is more similar to T. semblidis than to T. kurosuae in the proportion of flightless males. In addition, male wing polymorphisms of Trichogramma in relation to mating systems could also show a similarity between T. tajimaense and T. semblidis when considering both species as quasi‐gregarious parasitoids of Sialis eggs.     

Linkage mapping of the locus responsible for congenital multiple ocular defects in cattle on bovine Chromosome 18

Congenital multiple ocular defects (MOD) in Japanese black cattle is a hereditary ocular disorder with an autosomal recessive manner of inheritance, showing developmental defects of the lens, retina, and iris, persistent embryonic eye vascularization, and microphthalmia. In the present study, we mapped the locus responsible for the disorder by linkage analysis using 240 microsatellite markers covering the entire bovine genome and an inbred pedigree obtained from commercial herds. The linkage analysis demonstrated a significant linkage between the disorder locus and markers on the proximal region of bovine Chromosome (BTA) 18 with the maximum LOD score of 5.1. Homozygosity mapping using the haplotype of the linked markers further refined the critical region. The results revealed the localization of the locus responsible for MOD in an approximately 6.6-cM region of BTA18. Comparison of published linkage and radiation hybrid (RH) maps of BTA18 with its evolutionary ortholog, human Chromosome (HSA) 16, revealed several potential candidate genes for the disorder including the MAF and FOXC2 genes.     

Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.     

Novel contribution of cell surface and intracellular M1‐muscarinic acetylcholine receptors to synaptic plasticity in hippocampus

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.