Leak-free million-fold DNA amplification with locked nucleic acid and targeted hybridization in one pot.

An isothermal cascade reaction that exponentially amplifies pre-designed, single-stranded DNA as a sensor and signal amplifier module for DNA-based computing and molecular robotics was developed. Taking advantage of the finding that locked nucleic acid can suppress problematic ab initio DNA synthesis, up to million-fold amplification rates and concurrent hybridization were achieved at a physiological temperature in a single reactor. Although the effect of locked nucleic acid introduction to the templates was complicated, undesired leak DNA amplification was generally suppressed in the amplification reaction for distinct DNA sequences. The present reaction that senses one DNA as an input and generates a large amount of another DNA as an output, exhibiting a high correlation between the molecular concentration and the amplification time, is applicable for nucleic acid quantification.

Prognostic usefulness of arm circumference and nutritional screening tools in older patients with cardiovascular disease.

BACKGROUND AND AIM: Arm circumference (AC) and nutritional screening tools have been shown to have prognostic capability in patients with cardiovascular disease (CVD). This study aimed to compare the prognostic predictive capabilities of AC and nutritional screening tools in older patients with CVD. METHODS AND RESULTS: The study population consisted of 949 admitted patients ≥60 years old with CVD. Patients underwent AC measurement and nutritional screening before hospital discharge. We used the controlling nutritional status index (CONUT), the geriatric nutritional risk index (GNRI), and the prognostic nutritional index (PNI) as nutritional screening tools. The end point of the study was all-cause mortality. The mean age of the study population was 72.3 ± 7.2 years, and 68.2% of the patients were male. A total of 130 deaths occurred over a median follow-up period of 2.2 years (interquartile range, 1.1-3.8 years). After adjusting for other prognostic factors, AC (hazard ratio [HR]: 0.59; p < 0.001), CONUT (HR: 0.82; p = 0.016), GNRI (HR: 0.77; p = 0.040), and PNI (HR: 0.80; p = 0.014) were significant predictors of mortality. However, adding AC to the multivariate-adjusted model (0.739 vs. 0.714, respectively; p = 0.037), but not CONUT, GNRI, or PNI (0.724, 0.717, and 0.723 vs. 0.714; p = 0.072, p = 0.306, and p = 0.127, respectively), significantly increased the area under the curve on receiver operating characteristic curve. CONCLUSIONS: AC, but not nutritional screening tools, plays a complementary role to preexisting prognostic factors for predicting prognosis in older patients with CVD.

Foraging at the edge of chaos: internal clock versus external forcing.

Activity rhythms in animal groups arise both from external changes in the environment, as well as from internal group dynamics. These cycles are reminiscent of physical and chemical systems with quasiperiodic and even chaotic behavior resulting from "autocatalytic" mechanisms. We use nonlinear differential equations to model how the coupling between the self-excitatory interactions of individuals and external forcing can produce four different types of activity rhythms: quasiperiodic, chaotic, phase locked, and displaying over or under shooting. At the transition between quasiperiodic and chaotic regimes, activity cycles are asymmetrical, with rapid activity increases and slower decreases and a phase shift between external forcing and activity. We find similar activity patterns in ant colonies in response to varying temperature during the day. Thus foraging ants operate in a region of quasiperiodicity close to a cascade of transitions leading to chaos. The model suggests that a wide range of temporal structures and irregularities seen in the activity of animal and human groups might be accounted for by the coupling between collectively generated internal clocks and external forcings.

Symmetry breaking in escaping ants.

The phenomenon of herding is a very general feature of the collective behavior of many species in panic conditions, including humans. It has been predicted theoretically that panic-induced herding in individuals confined to a room can produce a nonsymmetrical use of two identical exit doors. Here we demonstrate the existence of that phenomenon in experiments, using ants as a model of pedestrians. We show that ants confined to a cell with two symmetrically located exits use both exits in approximately equal proportions to abandon it in normal conditions but prefer one of the exits if panic is created by adding a repellent fluid. In addition, we are able to reproduce the observed escape dynamics in detail using a modification of a previous theoretical model that includes herding associated with a panic parameter as a central ingredient. Our experimental results, combined with theoretical models, suggest that some features of the collective behavior of humans and ants can be quite similar when escaping under panic.

Avalanches in one-dimensional piles with different types of bases.

We perform a systematic experimental study of the influence of the type of base on the avalanche dynamics of slowly driven 1D ball piles. The control of base details allows us to explore a wide spectrum of pile structures and dynamics. The scaling properties of the observed avalanche distributions suggest that self-organized critical behavior is approached as the "base-induced" disorder at the pile profile increases.

Prospective study of mother-to-infant transmission of hepatitis C virus.

BACKGROUND: Mother-to-infant transmission of hepatitis C virus (HCV) could become the main route of HCV infection in the future because there are no methods available to prevent vertical infection. The aim of this study was to determine the incidence of mother-to-infant transmission in infants born to mothers who tested positive for anti-HCV antibodies and to elucidate associated risk factors for transmission. METHODS: Screening was conducted for 16,800 pregnant women with an anti-HCV antibodies test, and 154 mothers were positive. From the positive group 141 mothers were enrolled in the study and their 147 infants were followed from birth for serum alanine aminotransferase activity, anti-HCV antibodies and HCV RNA. HIV infection was tested in 73 of 141 mothers, all of whom were negative. RESULTS: Thirty-three infants were dropped from the study because they were followed for <6 months or were not tested adequately. Of the 114 infants finally evaluated 9 (7.8%) had detectable HCV RNA. The transmission rate was not influenced by the mode of delivery [vaginal delivery, 8 of 90 vs. cesarean section, 1 of 24 (P = 0.396)] or by the type of feeding [9 of 98 for breast-fed infants vs. 0 of 16 for formula-fed infants (P = 0.243)]. All infected infants were born to mothers who had HCV viremia at the delivery (P = 0.040) and to those with a high viral load (P = 0.019). CONCLUSIONS: Our prospective study showed that the transmission rate of mother-to-infant HCV infection was 7.8% in anti-HCV antibody-positive mothers. Risk was related to the presence of maternal HCV viremia at delivery and a high viral load in the mothers.

Role of BLNK in oxidative stress signaling in B cells.

BLNK (B cell linker protein) represents a central linker protein that bridges the B cell receptor-associated kinases with a multitude of signaling pathways. In this study, we have investigated the role of BLNK in oxidative stress signaling in B cells. H2O2 treatment of B cells induced a rapid tyrosine phosphorylation of BLNK in a H2O2 dose-dependent manner, which was inhibited in Syk-deficient DT40 cells. Calcium mobilization in BLNK-deficient as well as Syk-deficient and phospholipase C (PLC)-gamma2-deficient cells after H2O2 treatment was completely abolished. These were derived from decreased inositol 1,4,5-trisphosphate generation through PLC-gamma2 in BLNK-deficient cells. Moreover, viability of BLNK-deficient as well as PLC-gamma2-deficient cells after exposure to low doses of H2O2 was dramatically enhanced compared with that of the wild-type cells. Furthermore, c-Jun N-terminal kinase activation following high doses of H2O2 stimulation, but not low doses of H2O2 stimulation, was abrogated in BLNK-deficient as well as Syk-deficient cells. These findings have led to the suggestion that BLNK is required for coupling Syk to PLC-gamma2, thereby accelerating cell apoptosis in B cells exposed to low doses of H2O2.

Tissue distribution of constitutive proteasomes, immunoproteasomes, and PA28 in rats.

We investigated the expression of standard proteasomes, immunoproteasomes, and their regulators, PA28, and PA700, in rat tissues. Immunoproteasomes (with subunits LMP2, LMP7, and MECL1) were abundant in the spleen but almost absent in the brain. In contrast, standard proteasomes (with X, Y, and Z) were highly expressed in the brain but not in the spleen. Both proteasome types were present in the lung and the liver. PA700 subunits (p112, S5a, and p45) were found in all tissues. PA28alpha, PA28beta, and PA28gamma were also expressed in all tissues, except for the brain which contained very little PA28beta. The results did not depend on rat sex or age. The cleavage specificity for peptide substrates differed greatly between brain and spleen proteasomes. Hybrid proteasomes, containing both PA28alphabeta and PA700, were not present in the brain but in all other tissues examined.

Syk is required for the activation of Akt survival pathway in B cells exposed to oxidative stress.

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. Here we report that Syk is required for the activation of the phosphatidylinositol (PI) 3-kinase-Akt survival pathway in B cells exposed to oxidative stress. Phosphorylation and activation of the serine-threonine kinase Akt were markedly increased in B cells treated with H(2)O(2). In Syk-deficient DT40 cells treated with low doses of H(2)O(2) (10-100 microm), Akt activation was considerably reduced. Pretreatment with wortmannin, a PI 3-kinase-specific inhibitor, completely blocked the Syk-dependent Akt activation. Following stimulation by low doses of H(2)O(2), a significant increase in PI 3-kinase activity was found in wild-type but not in Syk-deficient cells. These findings suggest that PI 3-kinase mediates Syk-dependent Akt activation pathway. Furthermore, viability of Syk-deficient cells, after exposure to H(2)O(2), was dramatically decreased and caspase-9 activity was greatly increased compared with that of the wild-type cells. These results suggest that Syk is essential for the Akt survival pathway in B cells and enhances cellular resistance to oxidative stress-induced apoptosis.

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells.

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.

[Examination of soluble cell adhesion molecules in pregnancy induced hypertension].

This study was performed to determine whether or not the soluble-intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1) and endothelial leukocyte adhesion molecule-1 (sELAM-1) are sensitive markers of pregnancy induced hypertension (PIH). sICAM-1 concentrations were significantly higher (p < 0.05) in the mild PIH compared to non-pregnant women and normal pregnant groups. sVCAM-1 concentrations in the mild PIH group and the severe PIH group were significantly higher than the non-pregnant women group (p < 0.0001, p < 0.01, respectively) and the normal pregnant group (p < 0.0001, p < 0.0005, respectively). The concentrations of sELAM-1 in the mild PIH group were also significantly higher compared to normal pregnant group (p < 0.01). Our results suggest that soluble cell adhesion molecules may be useful markers detecting endothelial damage and dysfunction in patients with PIH.

Overtone Transitions of 12CO2 beyond the Venus Bands.

Combination overtone transitions of 12CO2 much weaker than the Venus bands in the near infrared (11 700-14 200 cm-1) have been observed for the first time by using photoacoustic spectroscopy. A total of six bands from two Fermi polyad groups were recorded and both the band origins and relative intensities have been determined and compared with the calculated values. Through a comparison of the relative absorption cross sections within the same type of polyads, intensity shifts have been observed and will be discussed. Copyright 1999 Academic Press.

[A case of congenital complete heart block in a mother with anti-52 kD SS-A/Ro antibodies].

We report a case of congenital complete heart block (CCHB). A 38-year-old woman was admitted our hospital because of fetal bradycardia at 21 weeks 3 days of gestational age. She had no symptom of collagen disease. On admission, laboratory data showed positive anti-nuclear antibodies, anti-SS-A/Ro antibodies and anti-52 kD SS-A/Ro antibodies. But anti-60 kD SS-A/Ro and anti-SS-B/La antibodies were negative. Consequently anti-52 kD SS-A/Ro antibodies positive woman had an infant with CCHB. The baby was equipped with pacemaker at the age of 2 months. This report suggests that anti-52 kD SS-A/Ro antibodies may play an important role in the development of CCHB.

CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome.

Isolation and characterization of a novel perchloric acid-soluble protein inhibiting cell-free protein synthesis.

We found a novel protein in the postmitochondria supernatant fraction of rat liver, which is soluble in 5% perchloric acid and strongly inhibits protein synthesis in a rabbit reticulocyte lysate system. The protein extracted from the supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The protein was shown to consist of two identical subunits with a molecular mass of 14 kDa. By immunoscreening with the rabbit antisera against the protein, a cDNA encoding the protein was cloned and sequenced. The cDNA contained an open reading frame of 411 base pairs encoding a 136-amino acid protein with a molecular mass of 14,149 Da. The deduced amino acid sequence was completely identical with that constructed from all of the above peptides. Interestingly, the perchloric acid-soluble protein inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner from RNase A. The protein is likely to inhibit an initiation stage of cell-free protein synthesis. Among the rat tissues tested, the protein was located only in liver and kidney. These findings are the first report on a new inhibitor that may be involved in the regulation of protein synthesis in those tissues.

Molecular cloning of two types of cDNA encoding subunit RC6-I of rat proteasomes.

A new subunit, named RC6-I, of the rat 20 S proteasome was purified and the partial amino acid sequences of several peptide fragments obtained by digestion with lysyl-endopeptidase were determined by Edman degradation. Amplification of cDNAs encoding RC6-I by the polymerase chain reaction (PCR) technique revealed two types of cDNA, tentatively designated as RC6-IL and RC6-IS in order of size. The nucleotide sequences of the two cDNAs are identical except that RC6-IL contains an insertion of 18 nucleotides in the coding region compared with RC6-IS. The polypeptide predicted from the open reading frame of RC6-IS cDNA consists of 248 amino acid residues with a calculated molecular weight of 27,783. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology analysis showed that RC6-I belongs to the alpha-type subfamily of the proteasome gene family, which shows similarity to the alpha-subunit of the archaebacterium Thermoplasma acidophilum proteasome, and that the 18 nucleotide insert, encoding six amino acid residues, VVASVS, appears to be unique to RC6-IL, because this motif has not been conserved in any other alpha-type subunit. By reverse transcription (RT)-PCR analysis, the mRNAs for both RC6-IL and RC6-IS were found in all the rat tissues examined. These results suggest that proteasomes are present as a heterogeneous population, possibly for acquisition of diversity of functions.

Primary structures of two homologous subunits of PA28, a gamma-interferon-inducible protein activator of the 20S proteasome.

The primary structures of two proteins that comprise PA28, an activator of the 20S proteasome, have been determined by cDNA cloning and sequencing. These protein subunits, termed PA28 alpha and PA28 beta, are about 50% identical to one another and are highly conserved between rat and human. PA28 alpha and PA28 beta are homologous to a previously described protein, Ki antigen, whose function is unknown. PA28 alpha, but neither PA28 beta nor Ki antigen, contains a 'KEKE motif', which has been postulated to promote the binding of proteins having this structural feature. PA28 alpha and PA28 beta were coordinately regulated by gamma-interferon, which greatly induced mRNA levels of both proteins in cultured cells. The mRNA level of the Ki antigen also increased in response to gamma-interferon treatment, but the magnitude of the increase was less than that for the PA28s, and the effect was transient. These results demonstrate the existence of a new protein family, at least two of whose members are involved in proteasome activation. They also provide the basis for future structure/function studies of PA28 subunits and the determination of their relative physiological roles in the regulation of proteasome activity.

cDNA cloning of a new putative ATPase subunit p45 of the human 26S proteasome, a homolog of yeast transcriptional factor Sug1p.

The nucleotide sequence of a cDNA that encodes a new regulatory subunit, named p45, of the 26S proteasome of human hepatoblastoma HepG2 cells has been determined. The polypeptide predicted from the open reading frame consists of 406 amino acid residues with a calculated molecular weight of 45770 and isoelectric point of 8.35. The sequences of several fragments of bovine p45, determined by protein chemical analyses, spanning 27% of the complete structure, were found to be in excellent accord with those deduced from the human cDNA sequence. Computer analysis showed that p45 belongs to a family of putative ATPases which includes regulatory components of 26S proteasomes. The overall structure of p45 was found to be homologous to that of yeast Sug1p, which has been identified as a transcriptional factor. It is closely similar, but not identical to the sequence reported for Trip1, a functional homolog of Sug1p in human tissues. These results are consistent with the possibility that Sug1-like proteins with distinct sequence function in transcription and protein degradation in human cells. However, the alternative hypothesis, that the same gene locus encodes both p45 and Trip1, cannot be excluded on the basis of such closely similar sequences. In either case, both proteins are likely to function equivalently well in either transcription or protein degradation.

Isolation and characterization of the HC8 subunit gene of the human proteasome.

For study of the molecular basis of regulation of proteasome gene expression, we isolated the gene encoding the alpha-type HC8 subunit of the human proteasome. About 2.3 kb of the 5' flanking region of this gene was tested for promoter function by chloramphenicol acetyltransferase assay. This analysis revealed that CAAT and TATA boxes, but not a GC box, are essential for its promoter activity. These results differed from previous findings that the genes for the alpha-type HC3 and beta-type HC5 subunits of the human proteasome have a TATA-less promoter and that two or three GC boxes function as the promoter sequences (Tamura, T. et al. (1994) J. Mol. Biol. 244, 1117-1124). We mapped the HC8 gene at q23 on human chromosome 14, which differs from the chromosomal locations of nine other proteasomal subunit genes mapped so far.