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PubMed2005

RNA interference of Arabidopsis beta-amylase8 prevents maltose accumulation upon cold shock and increases sensitivity of PSII photochemical efficiency to freezing stress.

It has been suggested that beta-amylase (BMY) induction during temperature stress in Arabidopsis could lead to starch-dependent maltose accumulation, and that maltose may contribute to protection of the electron transport chain and proteins in the chloroplast stroma during acute stress. A time-course transcript profiling analysis for cold shock at 4 degrees C revealed that BMY8 (At4g17090) was induced specifically in response to cold shock, while major induction was not observed for any of the other eight beta-amylases. A parallel metabolite-profiling analysis revealed a robust transient maltose accumulation during cold shock. BMY8 RNAi lines with lower BMY8 expression exhibited a starch-excess phenotype, and a dramatic decrease in maltose accumulation during a 6-h cold shock at 4 degrees C. The decreased maltose content was also accompanied by decreased glucose, fructose and sucrose content in the BMY8 RNAi plants, consistent with the roles of beta-amylase and maltose in transitory starch metabolism. BMY8 RNAi lines with reduced soluble sugar content exhibited diminished chlorophyll fluorescence as F(v)/F(m) ratio compared with wild type, suggesting that PSII photochemical efficiency was more sensitive to freezing stress. Together, carbohydrate analysis and freezing stress results of BMY8 RNAi lines indicate that increased maltose content, by itself or together through a maltose-dependent increase in other soluble sugars, contributes to the protection of the photosynthetic electron transport chain during freezing stress.

Arabidopsis↗
PubMed1993

N-terminal amino acid sequences of 440 kDa hemoglobins of the deep-sea tube worms, Lamellibrachia sp.1, Lamellibrachia sp.2 and slender vestimentifera gen. sp.1 evolutionary relationship with annelid hemoglobins.

The deep-sea tube worm Lamellibrachia, belonging to the phylum Vestimentifera, contains two types of extracellular hemoglobins, a 3,000 kDa hemoglobin and a 440 kDa hemoglobin. The latter hemoglobin is composed of four heme-containing chains with molecular masses of 16-18 kDa. We have collected Lamellibrachia sp.1, Lamellibrachia sp.2 and Slender vestimentifera gen. sp.1 from the deep-sea cold-seep or hydrothermal areas at a depth of 1100-1400 m. The four constituent chains of the 440 kDa hemoglobin were isolated from each of the three tube worms by reverse-phase chromatography, and the N-terminal amino acid sequences of 16-44 residues were determined by automated protein sequencer. The amino acid sequences of the homologous chains showed high homology (76-85%), suggesting that they are closely related. The sequences also showed 45-49% homology with annelid hemoglobins. A phylogenetic tree constructed from hemoglobin sequences showed that the tube worm Lamellibrachia, the polychaete Tylorrhynchus and the oligochaete Lumbricus diverged from a common ancestor at almost the same time, about 450 million years before present.

Amino Acid Sequence↗
PubMedDate not supplied

Reversible association of myosin with the platelet cytoskeleton.

Platelets circulating in the human blood stream are smooth disk-shaped structures. The disks change within seconds of exposure to ADP or thrombin to irregular spheres bearing filopodia and pseudopodia. It is well-established that platelets also change shape (although more slowly) when chilled to 5 degrees C and revert to disks on rewarming. This cold-induced shape change may be due to the depolymerization of the submembranous microtubule ring. However, we found that chilling in the presence of Taxol, which stabilizes the microtubules, still results in shape change. Chilled platelets show an increase in the amount of myosin in the Triton-X insoluble residue or 'cytoskeleton' which is correlated in time both with phosphorylation of the myosin regulatory light chain and with the induced shape change. We suggest here that the slow cold-induced change from disks to spheres is due primarily to a gradual activation of myosin.

Blood Platelets↗
PubMed1987

Prevention of the hand-arm vibration syndrome.

In order to determine the prevalence of the hand-arm vibration syndrome before preventive countermeasures were taken, a special health examination was carried out among 417 national forestry workers operating chain saws in the northern area of Hokkaido, Japan. Thirty-two chain-saw workers (7.7%) were diagnosed as exhibiting the vibration syndrome. The highest prevalence rates were 15.8% among the workers who had operated the saws for 11 to 15 years and 20.3% for workers in their 50s. Improved work conditions for chain-saw workers have increasingly prevented the vibration syndrome in the state forests of Japan since 1978. The present report covered the six years since 1978, and evaluated the effects of these improved work conditions on chain-saw workers from data on the recovery rates of skin temperature and the vibration sense threshold after a cold provocation test. As a result, recovery rates of skin temperature and the vibration sense thresholds at the fifth and tenth minutes after the immersion of the hands in cold water were significantly better than those six years earlier. It is suggested that adequate restrictions on the operating time of the chain saw and on the age of workers can completely prevent the vibration syndrome even if the total operating time is appreciably lengthened.

Adult↗
PubMed1992

Impact of specimen handling and storage on detection of hepatitis C virus RNA.

Direct detection of hepatitis C virus (HCV) RNA in serum or plasma is useful for validating the performance of anti-HCV assays and for the discrimination of persons with persistent HCV infections from those with resolved infections. Quantitation of HCV RNA may also be useful for disease prognosis and therapeutic monitoring. Previous studies have reported detection of HCV RNA in 50 to 70 percent of blood donors who were positive on anti-HCV supplemental tests. There is concern that specimen processing and storage conditions might influence the stability, and hence the detectability, of HCV RNA. To address this concern, the rate of detection of HCV RNA by the polymerase chain reaction (PCR) using donor pilot tube sera (PTS) previously subjected to routine donor screening and supplemental testing was compared with HCV PCR results obtained with fresh-frozen plasma (FFP) derived from the same donations. All 16 anti-HCV supplemental test-positive donations evaluated were HCV RNA positive with FFP, whereas only 10 (62.5%) were positive with PTS (p = 0.024). None of 11 FFP or PTS samples from HCV enzyme immunoassay-reactive donations not confirmed by supplemental anti-HCV assays tested positive for HCV RNA. Direct comparison of sample type (serum vs. plasma) and various storage conditions using specimens from two seropositive donors showed that room-temperature storage results in marked reduction in HCV RNA signal, while replicate freezing and thawing caused a moderate reduction. These data indicate that well-controlled sample processing and storage conditions are critical to the sensitive and potentially quantitative analysis of HCV RNA.

Blood Preservation↗
PubMed1989

[Analysis of subjective symptoms of coldness and numbness in the upper and lower limbs among patients with vibration syndrome].

Coldness and numbness of the lower limbs in patients with vibration syndrome were studied in correlation with the frequency of attacks of vibration-induced white finger (VWF), coldness and numbness of the upper limbs, and awakening at night due to numbness of the arm. Subjects were 229 patients aged 50-69 years without disease other than vibration syndrome who had mainly operated chain saws. The present study showed that patients with more frequent VWF attacks had a higher prevalence and frequency of coldness in the fingers, awakening at night due to numbness of the arm, and coldness and numbness in the legs. Particularly patients having almost daily attacks of VWF had a high prevalence of these symptoms. Positive correlation was observed in prevalence and frequency between coldness of the fingers and that of the legs, and between coldness and numbness in the legs. The prevalence of VWF, numbness and coldness of the fingers, and coldness of the legs was higher the longer the total chain saw operating period. The present findings suggest that patients with severe circulatory disturbances in the upper limbs have severe circulatory disturbance in the lower limbs. Further studies on circulatory disturbances in the lower limbs are indicated, and their mechanism involved should be also pursued from the pathogenetic point of view.

Aged↗
PubMed1986

Hemolytic anemia due to a mixture of low-titer IgG lambda and IgM lambda agglutinins reacting optimally at 22 degrees C.

A 65-year-old white man had severe hemolytic anemia due to a mixture of low-titer IgG lambda and IgM lambda agglutinins showing optimum reactivity at 22 degrees C. The IgG agglutinins were detected by manual indirect antiglobulin test (IAT) using anti-IgG, and had a titer of 1 at 37 degrees C, 128 at 22 degrees C and 16 at 4 degrees C against adult type O red blood cells (RBC). The corresponding titers with cord RBC were 1 (37 degrees C), 64 (22 degrees C) and 8 (4 degrees C). Proteolytic enzyme and neuraminidase treatment of RBC did not decrease these titers. No known specificity could be assigned to these agglutinins. The isolated agglutinins (recovered by cold adsorption, warm elution) were shown by immunoelectrophoresis to be IgG lambda antibodies. They did not bind complement in vitro, consistent with the finding that the patient had negative manual direct antiglobulin test (DAT) by anti-C3d. It could be shown only by automated IAT that patient's serum also contained IgM cold agglutinins which also reacted best at 22 degrees C and appeared to be of lambda light-chain type. The patient responded to corticosteroid therapy and remains well without treatment 14 months after the hemolytic episode. The presence of IgG cold agglutinins may be predictive of a favorable response to corticosteroid therapy.

Aged↗
PubMed1998

Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp.

The psychorotrophic Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5 degrees C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C10 to C21 alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5 degrees C. Mineralization of hexadecane at 5 degrees C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction-gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis the A gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25 degrees C.

Alkanes↗
PubMed1986

The V kappa IIIb light chain sub-subgroup. II. Isotype expression in acquired hypogammaglobulinaemia and as a function of age.

The V kappa IIIb sub-subgroup of the human immunoglobulin V kappa III light chain subgroup has at least two unique properties. First, some IgM monoclonal autoantibodies, in particular cryoprecipitable anti-IgG (Kunkel et al., 1974; Ledford et al., 1983), anti-low density lipoprotein (Ledford et al., 1983) and certain cold agglutinin (Feizi et al., 1976) antibodies predominantly contain V kappa IIIb light chains. Second, in normal adult human serum, V kappa IIIb comprises approximately 25% of the IgM but less than 0.4% of the IgG or IgA kappa-chain pools (Moynihan, Looney & Abraham, 1985). Studies have suggested an altered regulation of IgM, IgG, and IgA-V kappa IIIb in some patients with acquired hypogammaglobulinaemia, a heterogeneous group of immunodeficiencies with low serum levels of Ig. An increase in the serum levels of V kappa IIIb light chains has been noted in some of these patients (Solomon & Mclaughlin, 1969) which is apparently due to elevated levels of light chains of the V kappa IIIb sub-subgroup (Greenstein & Abraham, 1984). However, the isotype association of V kappa IIIb in these patients has not been determined. In order to clarify whether the noted increase in V kappa IIIb levels is due to its selective expression with IgM or to an abnormality of immunoregulation, the heavy chain isotypes associated with V kappa IIIb light chains have been determined in a previously studied group of adults with acquired hypogammaglobulinaemia (Greenstein & Abraham, 1984). Further, the isotype distribution of V kappa IIIb light chains in another group of functional, albeit transient, hypogammaglobulinaemics (i.e. normal neonates) has also been studied by measuring IgM-V kappa IIIb levels in cord blood, and compared to the V kappa IIIb serum levels found in aged adults (mean age 75 years).

Adult↗
PubMed1998

Comparative effects of University of Wisconsin and Euro-Collins solutions on pulmonary mitochondrial function after ischemia and reperfusion.

BACKGROUND: The aim of this study was to compare the effects of Euro-Collins and University of Wisconsin solutions on pulmonary mitochondrial function after cold ischemia and subsequent warm reperfusion. METHODS: Seventeen pigs underwent lung harvesting after classical lung flush with either University of Wisconsin or Euro-Collins solutions. The mitochondria were isolated from fresh swine lungs, from swine lungs subjected to 24 hr of cold ischemia, and from swine lungs subjected to 24 hr of ischemia followed by 30 min of subsequent ex vivo reperfusion at 37 degrees C with Krebs-Henseleit buffer solution and air ventilation. Mitochondrial oxidative phosphorylation parameters were determined in isolated mitochondria by in vitro measurement of oxygen consumption rates. During reperfusion, the lung function was assessed by the pulmonary aerodynamic parameters and the pulmonary vascular resistance. RESULTS: Relative to controls, mitochondria submitted to cold ischemia showed an alteration in the oxidoreductase activities of the respiratory chain. However, the yield of oxidative phosphorylation was conserved. After reperfusion, pulmonary mitochondria underwent a significant worsening in the oxidoreductase activities of the respiratory chain, and a decrease in the respiratory control and the efficiency of oxidative phosphorylation. Meanwhile, the reperfused lungs showed evidence of early dysfunction, assessed by the aerodynamic parameters and pulmonary vascular resistance. In this model, there was no advantage of University of Wisconsin solution over Euro-Collins solution. CONCLUSIONS: The mild mitochondrial alterations after cold ischemia were not sufficient to explain the limited tolerance of lung to ischemia. After reperfusion, the mitochondrial damage was more severe and could be involved in the posttransplant lung dysfunction.

Adenosine↗
PubMed2004

Mobilization of opioid-containing polymorphonuclear cells by hematopoietic growth factors and influence on inflammatory pain.

BACKGROUND: Leukocytes can control inflammatory pain by secretion of opioid peptides, stimulated by cold-water swimming or local injection of corticotropin-releasing factor, and subsequent activation of opioid receptors on peripheral sensory neurons. This study investigated whether mobilization of polymorphonuclear cells (PMN) by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) enhances immigration of opioid-containing PMN and peripheral opioid analgesia in rats with Freund complete adjuvant-induced hind paw inflammation. METHODS: In circulating PMN of rats treated with G-CSF+SCF and sham-treated rats, opioid peptide content was measured by radioimmunoassay. Expression of adhesion molecules (CD62L, CD49d, CD18), in vitro migration in the Boyden chamber, and infiltrating leukocytes were analyzed by flow cytometry. Chemokine messenger RNA transcription was quantified by LightCycler polymerase chain reaction. Paw pressure threshold was measured at baseline, after cold-water swimming, and after injection of corticotropin-releasing factor. RESULTS: G-CSF+SCF treatment increased circulating PMN (11-fold, P < 0.05). Mobilized PMN had decreased content of beta-endorphin but not of Met-enkephalin per cell, down-regulation of CD62L, up-regulation of CD49d (but no change in CD18), and reduced migration toward higher chemokine concentrations (all P < 0.05). In the paw, one of four chemokine messenger RNAs was significantly expressed during the first 2 h of inflammation (P < 0.05), immigration of PMN and opioid-containing cells was slightly increased (1.5-fold, P < 0.05), and baseline paw pressure threshold, as well as paw pressure threshold increases induced by corticotropin-releasing factor and cold-water swimming, were unchanged (P > 0.05). CONCLUSIONS: G-CSF+SCF mobilized circulating opioid-containing PMN but had a minor influence on cell migration and peripheral analgesia, probably because of the low expression of chemokines in the inflamed paw and one of the decreased beta-endorphin content in PMN.

Animals↗
PubMed1982

Microperoxisomes and mitochondria of brown adipose tissue. Hydrodynamic parameters, isolation and capacity of long-chain fatty acid oxidation.

1. Analytical differential centrifugation of brown adipose tissue homogenates from cold-acclimated guinea pigs revealed a polydispersity of both mitochondria and peroxisomes, with at least two populations of each organelle. The estimated values were sH = 16685+/-4220 S and sL = 4792+/-951 S (mitochondria), and sH = 3364+/-1706 S and sL = 889+/-177 S (peroxisomes). Based on these s values, an optimal procedure is described for the isolation of subcellular fractions enriched in mitochondria and peroxisomes, respectively. 2. When the mitochondrial and peroxisomal fractions were subjected to isopycnic gradient centrifugation on a self-generating gradient of poly(vinylpyrrolidone)-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of about 1.11 g/cm3 (main fraction of mitochondria) and 1.07 g/cm3 (main fraction of peroxisomes) were obtained. A value of 1.06 g/cm3 was found for the microsomal fraction. 3. The main peroxisomal fraction, isolated by gradient centrifugation, did not reveal any significant oxidation of palmitoyl-CoA as measured by conventional polarographic technique, whereas a small rate of oxidation (about 2.7+/-0.2 nmol/min per mg peroxisomal protein) was observed when measured as NAD+ reduction. This rate contributes no more than 1% of the mitochondrial oxidation of this fatty acid and it is, therefore, concluded that peroxisomal oxidation of the predominant long-chain fatty acids found in this tissue does not make a quantitatively significant contribution to fatty acid oxidation.

Acyl Coenzyme A↗
PubMed1977

Hemoglobin Tarrant: alpha126(H9) Asp leads to Asn. A new hemoglobin variant in the alpha1beta1 contact region showing high oxygen affinity and reduced cooperativity.

Hemoglobin (Hb) Tarrant was detected by its electrophoretic mobility on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). On cellulose acetate it moved as a band between hemoglobins F and S, and on citrate agar as a band at hemoglobin S. The test for solubility in 2 M phosphate buffer with Na2S2O4 was negative. The new variant has a substitution of asparagine for aspartic acid in position 126 of the alpha-chain, one of the sites involved in the alpha1beta1 contact. Furthermore, in deoxyhemoglobin aspartic acid 126 of each alpha chain also forms a non-covalent electrostatic salt bridge with arginine 141 of the corresponding alpha chain (Perutz, M. F. and Ten Eyck, L. F. (1972) Cold Spring Harbor Symp. Quant. Biol. 36, 295-310 and Perutz, M. F. (1970) Nature 228, 726-739). As a consequence of this substitution in hemoglobin Tarrant, the deoxy conformation or T state is destabilized because these two bridges cannot be formed. This condition is reflected in high oxygen affinity and low cooperativity.

Amino Acid Sequence↗
PubMed1978

Effects of low temperature on in vivo and in vitro protein synthesis in Escherichia coli and Pseudomonas fluorescens.

The effects of temperature on protein synthesis by Escherichia coli, a mesophile, and Pseudomonas fluorescens, a psychotroph, were investigated by using whole-cell and cell extract preparations. After shifts to 5 degrees C, protein was synthesized at a slowly decreasing rate for 1 h by both organisms, after which P. fluorescens synthesized protein at a new rate corresponding to its 5 degrees growth rate, in contrast to E. coli which did not synthesize protein at a measurable rate. In vitro protein-synthesizing systems using MS-2 RNA, endogenous mRNA, and purified polysomes were utilized to investigate initiation of translation at 5 degrees C. In these systems, P. fluorescens cell extracts synthesized protein at linear rates for up to 2 h at 5 degrees C, whereas E. coli cell extracts synthesized protein for only 25 min at 5 degrees C. The rates of polypeptide elongation, as tested by the incorporation of phenylalanine into polyphenylalanine by cell extract protein-synthesizing systems from both organisms, were identical over the range of 25 to 0 degrees C. The polysome profiles of E. coli whole cells shifted from 37 to 5 degrees C showed accumulation of 70S ribosomal particles and ribosomal subunits at the expense of polysomes. Similar experiements done with P. fluorescens resulted in polysome reformation at 5 degrees C. In vitro experiments demonstrated that the 70S ribosomal particles, which accumulated in E. coli at 5 degrees C, were capable of synthesizing protein in vitro in the absence of added mRNA. These in vivo and in vitro results suggest that incubation of E. coli at subminimal temperatures results in a block in initiation of translation causing polysomal runoff and the accumulation of 70S particles, some of which are 70S monosomes.

Bacterial Proteins↗
PubMed1990

Functional expression of microsomal and mitochondrial cytochrome P-450 (d and SCC) in COS-7 cells from cloned cDNA.

Using full length cDNA introduced into COS-7 cells, two species of P-450 with entirely different physiological functions have been expressed in enzymatically active form. One is P-450d, which is known to reside in the microsomes of rat hepatocytes where it acts as a drug-metabolizing enzyme; the other is P-450(SCC), which catalyzes the conversion of cholesterol to pregnenolone in the rate-limiting reaction of steroidogenesis in mitochondria of adrenal cortex cells. Northern blot and immunoblot analyses revealed that the mRNA and protein of these P-450 species were efficiently produced in COS-7 cells. The protein contents amounted to nearly 0.1% of the total cell protein as estimated from immunoblotting and low temperature CO difference spectra. The subcellular localization of the products indicated that they were correctly sorted to the microsomes and mitochondria, respectively. We have succeeded in eliciting most of the activity of the expressed microsomal P-450d by reconstruction with NADPH-cytochrome P-450 reductase, while the optimal conditions for the mitochondrial enzyme in the COS cells remain to be studied. These results show the applicability of the COS-7 expression system to investigations of the functions of members of the P-450 superfamily whose cDNA has been newly isolated.

Animals↗
PubMed1994

Pathophysiology and clinical picture of hand-arm vibration syndrome in Japanese workers.

Hand-arm vibration syndrome is an occupational disease induced by long-term use of vibratory tools such as rock drills and chain saws. The three major stressors of vibration, noise and cold may produce various symptoms and signs not only with peripheral circulatory, nervous and muscle-joint disorders but also with general disorders. It is a point of controversy whether the symptoms and signs should be limited in the peripheral disorders. The question may involve differences in definition: vibration alone or work mode with vibratory tools. There are two viewpoints in the staging: peripheral and general viewpoints. The key concept in the peripheral viewpoint staging is a checkup to find disorders at the early stage and classify the peripheral disorders of the digits in detail. The general viewpoint staging seeks to grasp the general pictures of patients, and to classify from light to severe cases in the treatment. Clinical manifestations may include the general disorders in proportion to the severity of the syndrome according to our clinical experience. A differential diagnosis should be carefully made in the light of legal, medical and economic compensation. The treatments that we have used for approximately 20 years have beneficial effects on the whole-body, which include 1) physiobalneotherapy (therapeutic exercise, exercise in a pool and physiotherapy), 2) drug therapy (vasodilating drugs, autonomic stabilizers, etc.), 3) nerve blocking therapy, 4) surgical therapy for ulnar nerve paralysis or paresis, and 5) education for patients. Even with these therapies, a beneficial effect may not be observed in a short period. The recovery may be slow.(ABSTRACT TRUNCATED AT 250 WORDS)

Arm↗