Circadian rhythm of atrioventricular conduction predicts long-term survival in patients with chronic atrial fibrillation

The R-R interval of the electrocardiogram during atrial fibrillation (AF) appears absolutely irregular. However, the Poincaré plot of the R-R interval reveals a sector shape of distribution that is unique to AF. Furthermore, the height of lower envelope (LE1.0) of the distribution and the degree of scatter above the envelope (scattering index) may reflect the refractoriness and concealment of atrioventricular (AV) conduction, respectively. We previously observed that both the LE1.0 and scattering index show clear circadian rhythms in patients with chronic AF and that the rhythms are blunted in those with congestive heart failure and chronic AF. In the present study, we examined if the blunted circadian rhythm of the AV conduction has prognostic value in patients with chronic AF. We studied a retrospective cohort of 120 patients who underwent 24h Holter monitoring at baseline. During an observation period of 33 +/- 16 mon, there were 25 deaths (21%) including 13 cardiac and 8 stroke deaths. All patients showed significant circadian rhythms in both LE1.0 and scattering index with acrophases occurring at night; however, patients dying subsequently from cardiac causes, but not those from fatal stroke were blunted in the circadian rhythms (the amplitudes were < 55% of those in surviving patients). Furthermore, the reduced circadian amplitude of scattering index was an increased risk for cardiac death even after adjustment of coexisting cardiovascular risks [adjusted relative risk (95% confidence interval) per 1-SD decrement, 4.24 (1.54-11.6)]. When patients were divided by the circadian amplitude of the scattering index of 36.5 msec (mean minus 1-SD), the 5yr cardiac mortality below and above the cutoff was 57 and 6%, respectively (log-rank test, p < 0.001). We conclude that the blunted circadian rhythm of AV conduction is an independent risk for cardiac death in patients with chronic AF.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity

Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.     

Distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, in pulmonate snails

The distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, were examined in the nervous system of pulmonate snails. We chemically identified the authentic NdWFamide from a land snail (Euhadra congenita) and a freshwater snail (Lymnaea stagnalis). NdWFamide potentiated the heartbeat of those snails. Immunohistochemistry using anti-NdWFamide antibody demonstrated the distribution of NdWFamide-containing neurons and fibers in the central nervous system, as well as peripheral tissues, such as the cardiovascular region and accessory sex organs. These results suggest that NdWFamide is a neuropeptide mediating the neural regulation of the activity of the cardiovascular and reproductive systems of snails.     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

Membrane transduction pathway in the neuronal control of protein secretion by the albumen gland in Helisoma (Mollusca)

The albumen gland in Helisoma secretes a perivitelline fluid which surrounds each egg and is made up of several 66 kDa protein subunits and polysaccharide complexes. Forskolin, an adenylate cyclase activator, stimulated the secretion and release of the perivitelline fluid. An acidic extract of the central nervous system increased the intracellular concentration of cAMP in the albumen gland and this results in the release of the 66 kDa molecule and other proteins. Digestion of the brain extract with proteases abolished this activity, suggesting that the factor is a peptide. Cyclic AMP analogues and [BMX also stimulated the protein secretion in dose-dependent manner. Forskolin when added with the brain factor had an additive response. SQ22536, a non-competitive inhibitor of adenylate cyclase, inhibited brain extract dependent adenylate cyclase activity whereas aluminum fluoride, a G protein activator, was found to stimulate adenylate cyclase. Dopamine also stimulates protein secretion by the albumen gland and through the application of various agonists and antagonists of dopamine, it was established that the neurotransmitter acts via D1-like receptors by stimulating adenylate cyclase.     

Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization

Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid species.