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PubMed1998

Intragenic suppressors of Dictyostelium myosin G680 mutants demarcate discrete structural elements. Implications for conformational states of the motor.

We are using intragenic suppression to generate inferences about currently inaccessible conformations of the molecular motor myosin. We identified 19 unique suppressors of Dictyostelium G680A and G680V mutants and find that they are structurally and chemically restricted, suggestive of specific, compensatory mechanisms of action. Suppressors cluster in two adjacent elements of the myosin structure, one of which is dominated by substitutions that increase the volume of hydrophobic residues. The suppressors outline a general model for the mechanism of suppression of the G680A and G680V alterations. Secondary phenotypes conferred by suppressors independent of the G680 changes demonstrate that they will be useful substrates for biochemical and genetic characterization.

Animals↗
PubMed2005

Flavobacterium fryxellicola sp. nov. and Flavobacterium psychrolimnae sp. nov., novel psychrophilic bacteria isolated from microbial mats in Antarctic lakes.

Taxonomic studies were performed on seven strains isolated from microbial mats in Antarctic lakes of the McMurdo Dry Valleys. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains are related to the genus Flavobacterium; sequence similarity values with their nearest phylogenetic neighbours ranged from 97.0 to 98.7 %. Results of DNA-DNA hybridization and comparison of repetitive extragenic palindromic DNA-PCR fingerprinting patterns revealed that these strains are members of two distinct species. Genotypic results, together with phenotypic characteristics, allowed these species to be differentiated from related recognized Flavobacterium species. The isolates are psychrophilic, Gram-negative, chemoheterotrophic, rod-shaped cells. Their whole-cell fatty acid profiles are similar and include C(15 : 0), C(15 : 0) anteiso, C(15 : 0) iso, C(15 : 1)omega6c, C(16 : 0) iso, C(16 : 0) iso 3-OH, C(16 : 1) iso and summed feature 3 (which comprises C(15 : 0) iso 2-OH and/or C(16 : 1)omega7c) as the major fatty acid components. On the basis of these results, two novel species are proposed: Flavobacterium fryxellicola sp. nov., consisting of three strains with LMG 22022(T) (=CIP 108325(T)) as the type strain; and Flavobacterium psychrolimnae sp. nov., consisting of four strains with LMG 22018(T) (=CIP 108326(T)) as the type strain. DNA G+C contents of F. fryxellicola and F. psychrolimnae are 35.2-35.9 and 33.8-34.5 mol%, respectively.

Antarctic Regions↗
PubMed1994

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.

Amino Acid Sequence↗
PubMed1984

Immunochemical studies on an IgG lambda cryoglobulin in cold-induced urticaria.

A type I cryoglobulinaemia associated with cold-induced urticaria was demonstrated in a 64-year-old woman without primary disease. The cryoglobulin contained only IgG lambda as disclosed by immunofixation technique. Different physicochemical studies indicated that the IgG lambda component was monomeric at temperatures above 35 degrees C, but became polymerized below 35 degrees C. In addition crossed immunoelectrophoresis of plasma fibronectin from the patient showed a heterogeneous precipitate at low temperatures but a homogeneous precipitate at 25 degrees C indicating a complex formation at low temperature between IgG lambda and fibronectin. Fibronectin, however, was not essential for the cold precipitation of the cryoglobulin. The precipitation phenomenon at low temperatures was found to be a result of the physicochemical properties of the cryoglobulin itself unrelated to the antibody specificities tested. The importance of performing the immunochemical and physicochemical techniques at low temperature (7 degrees C) and at high temperature (35 degrees C) to gain knowledge of the nature of the protein, is emphasized. We conclude that only results obtained by relevant laboratory procedures might lead to correct classification and understanding of cryoglobulinaemia.

Aged↗
PubMed1999

A cold shock-induced cyanobacterial RNA helicase.

The ability to modify RNA secondary structure is crucial for numerous cellular processes. We have characterized two RNA helicase genes, crhB and crhC, which are differentially expressed in the cyanobacterium Anabaena sp. strain PCC 7120. crhC transcription is limited specifically to cold shock conditions while crhB is expressed under a variety of conditions, including enhanced expression in the cold. This implies that both RNA helicases are involved in the cold acclimation process in cyanobacteria; however, they presumably perform different roles in this adaptation. Although both CrhB and CrhC belong to the DEAD box subfamily of RNA helicases, CrhC encodes a novel RNA helicase, as the highly conserved SAT motif is modified to FAT. This alteration may affect CrhC function and its association with specific RNA targets and/or accessory proteins, interactions required for cold acclimation. Primer extension and analysis of the 5' untranslated region of crhC revealed the transcriptional start site, as well as a number of putative cold shock-responsive elements. The potential role(s) performed by RNA helicases in the acclimation of cyanobacteria to cold shock is discussed.

Amino Acid Sequence↗
PubMed1997

Rhinovirus stimulation of interleukin-8 in vivo and in vitro: role of NF-kappaB.

Neutrophil infiltration is a well-documented early event in the pathogenesis of rhinovirus (RV) infections. To further understand the mechanisms responsible for this neutrophilia, we determined whether interleukin (IL)-8 was present at sites of experimental RV infection in vivo and characterized the mechanism(s) by which RV stimulates IL-8 production in vitro. IL-8 was readily detectable in the nasal washings of all normal volunteers and did not increase with sham nasal inoculation. In contrast, RV infection caused a significant additional increase in nasal IL-8, the levels of which peaked 48-72 h after virus inoculation. RV was a potent stimulator of IL-8 protein production by A549 epithelial-like cells, MRC-5 fibroblasts, and normal human bronchial epithelial cells in vitro. This induction was associated with a significant increase in IL-8 mRNA accumulation and gene transcription. RV also stimulated IL-8 promoter-driven luciferase activity. This stimulation was significantly decreased by mutation of the nuclear factor (NF)-IL-6 site and was completely abrogated by mutation of the NF-kappaB site in this promoter. In addition, NF-kappaB-DNA binding activity was rapidly induced in RV-infected cells. This inducible binding was made up of p65 and, to a lesser extent, p50 NF-kappaB moieties. These studies demonstrate that IL-8 is present in normal nasal secretions and that the levels of IL-8 are further increased after RV infection. They also demonstrate that RVs are potent stimulators of IL-8 production and that this induction is mediated, at least in part, by an NF-kappaB-dependent transcriptional activation pathway. IL-8 may contribute to the pathogenesis of RV infection, and NF-kappaB activation may be a central event in RV-induced pathologies.

Adult↗
PubMed1997

Relationship between cold tolerance and generation of suppressor macrophages during acute cold stress.

Acute cold stress induces suppressor macrophages expressing large numbers of receptors to the crystallizable fragment (Fc) portion of immunoglobulin G (MAC-1+ FcgammaRII/IIIbright cells), resulting in the immunosuppression of splenocyte mitogenesis. The generation of MAC-1+ FcgammaRII/IIIbright cells is mediated by the action of glucocorticoids (GCs) through the GC-receptor. In the present study, the generation of MAC-1+ FcgammaRII/IIIbright cells in peritoneal exudate cells was closely related to the decrease of rectal temperature during 3-day exposure to 5 degrees C. We next investigated the effects of improved cold tolerance on the generation of MAC-1+ FcgammaRII/IIIbright cells during acute cold stress. Mice were adapted to cold by exposure to 5 degrees C for 3 wk (cold-acclimated mice) and then reexposed to 5 degrees C for 3 h (acute cold stress) after living at 25 degrees C for 24 h. The rectal temperature of cold-acclimated mice was not decreased by the acute cold stress. In addition, the proportion of MAC-1+ FcgammaRII/IIIbright cells in peritoneal exudate cell population from cold-acclimated mice was unaffected by the acute cold stress. The cold acclimation significantly attenuated the increases in serum corticosterone levels and the expression of the GC-receptor mRNA on peritoneal exudate cells in response to acute cold stress. These results suggest that the altered GC response to acute cold stress by the improvement of cold tolerance inhibits the generation of suppressor macrophages during acute cold stress.

Acclimatization↗
PubMed1999

Serotonin-induced coronary contraction increases after blood cardioplegia-reperfusion: role of COX-2 expression.

BACKGROUND: Coronary contraction has been implicated in causing suboptimal myocardial function after coronary bypass surgery. Addition of blood to cardioplegic solutions has been shown to improve endothelial function after cardioplegia. In this study, the effects of blood cardioplegia and brief reperfusion on vascular reactivity in patients with coronary artery disease and the expression (mRNA and protein) of enzymes involved in vasomotor regulation were examined. METHODS AND RESULTS: The atrial appendages of patients undergoing coronary artery surgery were harvested before cardiopulmonary bypass (control, n=8) and after bypass from a nonischemic tissue atrial segment exposed to cold, hyperkalemic blood cardioplegia (mean, 60 minutes) and a brief period (10 minutes) of reperfusion (CP-Rep, n=8). Responses of atrial arterioles were studied in vitro with video-microscopy. Reverse-transcriptase polymerase chain reaction and Western blotting were used to examine the expressions and protein content, respectively, of enzymes involved in vasomotor regulation. Serotonin caused a minimal dilation under baseline conditions but after CP-Rep elicited a potent contractile response that was inhibited in the presence of the selective inducible cyclooxygenase (COX-2) inhibitor NS398. Substance P caused an endothelium-dependent relaxation of atrial arterioles through release of nitric oxide, and ADP caused relaxation mediated through release of prostaglandins. After CP-Rep, relaxation to substance P was impaired, whereas endothelium-independent relaxation to nitroprusside and response to ADP were unchanged. Expression and protein level of COX-2 were significantly increased after CP-Rep. In contrast, expression of inducible (nitric oxide synthase-2) or constitutive endothelial (nitric oxide synthase-3) nitric oxide synthase, prostacyclin synthase, and constitutive cyclooxygenase (COX-1) were not altered after CP-Rep. CONCLUSIONS: CP-Rep increases serotonin-induced contraction of human microvessels caused by the release of products of COX-2 and the impaired release of nitric oxide. These findings have implications regarding altered coronary microvascular regulation and the cause of coronary spasm after cardiac surgery.

Aged↗
PubMed2003

Generation and characterization of an H9N2 cold-adapted reassortant as a vaccine candidate.

H9N2 subtype avian influenza viruses have been identified in avian species worldwide, and infections in pigs were confirmed in Hong Kong in 1998. Subsequently, H9N2 viruses were isolated from two children in Hong Kong in 1999, and five human infections were reported from China, raising the possibility that H9N2 viruses pose a potential pandemic threat for humans. These events prompted us to develop a vaccine candidate to protect humans against this subtype of influenza A viruses. Reassortant H1N1 and H3N2 human influenza A viruses with the six internal gene segments of A/Ann Arbor/6/60 (H2N2)(AA) cold-adapted (ca) virus have been tested extensively in humans and have proved to be attenuated and safe as live virus vaccines. Using classical genetic reassortment, we generated a reassortant that contains the hemagglutinin and neuraminidase genes from A/chicken/Hong Kong/G9/97 (H9N2) and six internal gene segments from the AAca virus. The G9/AAca reassortant virus exhibits the ca phenotype and the temperature-sensitive phenotypes of the AAca virus and was attenuated in mice. The reassortant virus was immunogenic and protected mice from wild-type H9N2 virus challenge. The G9/AAca virus bears the in vitro and in vivo phenotypes specified by the AAca virus and will be evaluated as a potential vaccine candidate in humans.

Acclimatization↗
PubMed1980

[Anatomo-pathological study of the evolution of cryonecrosis in the rat pancreas].

The progress of a cryonecrosis site occupying about one-third of the rat pancreas and induced with liquid nitrogen applied via a 10 mm luminal diameter probe applied for 2 min was followed. The lesion was not responsible for irreversible functional and anatomical lesions. Blood sugar remained within normal limits. Blood amylase peaked on the 1st day and was normal by the 14th. The treated area shrank considerably and was replaced by fibrous tissue within 30 days. The histological picture was similar to that encountered pathologically in man. Neither chain-type autolysis nor pseudocysts were observed.

Animals↗
PubMed1994

Sympathetic responses to hand-arm vibration and symptoms of the foot.

Vibration syndrome is generally considered to consist of disorders in the upper extremities which are directly exposed to vibration. However, it has been shown that vibration syndrome patients have circulatory disturbances in the foot as well: several chain-saw operators, who were little exposed to vibration of the foot, had Raynaud's phenomenon of the toes, and those with frequent attacks of vibration-induced white finger (VWF) had a higher prevalence of coldness felt in both the hands and the feet, and the patients with VWF had lower skin temperature of the toes as well as the fingers. Moreover, arterial pathological changes like medial muscular hypertrophy have been observed in both the fingers and the toes of the patients. Hand-arm vibration elicits a sympathetic nervous reflex, leading to vasoconstriction of the four extremities. Long-term repeated vasoconstriction may result in such arterial changes and then circulatory disturbances of the feet.

Arm↗
PubMed1975

A common idiotype on human macroglobulins with anti-I and anti-i specificity.

The reactions of an idiotypic antiserum prepared against an anti-I cold agglutinin (Da) were studied in radioimmunoassays which were designed to detect partial rather than complete idiotypic cross-reactions among cold agglutinins with anti-I and anti-i activity. Proteins showing strong idiotypic cross-reactions by gel precipitation could not inhibit the binding of 125I-labelled Da to anti-Da (individual specificity). It was necessary to use 125I-labelled cross-reactive proteins in order to demonstrate cross-idiotypic specificity and even the choice of the reference cross-reactive protein was important. The use of the 125I-labelled Fab fragment of a cross-reactive anti-I cold agglutinin (Low) allowed the detection of an idiotypic determinant common to at least 73% of cold agglutinins with specificity for the I-i antigen complex. Among these were two transiently occurring cold agglutinins following M. pneumoniae infection and infectious mononucleosis. This idiotypic determinant was not detected on cold agglutinins with anti-Pr specificity or on isolated monoclonal IgM or IgG lacking cold agglutinin activity, but it was present in low concentrations in pooled IgG. The common idiotypic determinant was found on IgM kappa cold agglutinins belonging to different VK subgroups and on an IgM lambda protein. A knowledge of the structural basis of such idiotypic cross-reactions among proteins from unrelated individuals should provide important information about the genes controlling the synthesis of site-related regions of antibodies.

Agglutinins↗
PubMed1993

[Immunoglobulins in hematology. Peripheral neuropathy associated with monoclonal IgM].

Peripheral neuropathy is often associated with a monoclonal IgM and occurs in about 3% of authentic Waldenström macroglobulinemias. In at least as many cases, the neuropathy is associated with a monoclonal IgM although lymphoplasmocyte proliferation is not detected, at least initially. In about 2/3 of the cases, the monoclonal IgM is a specific antibody against a myelin associated glycoprotein (MAG), two peripheral nerve glycolipids (SGPG and SGLPG), the peripheral nerve protein P0, other smaller molecular weight molecules of the peripheral nerves, or finally certain adhesion proteins such as N-CAM. All these IgM have a fine specificity against an epitope of the glucuronyl sulfate determinant present on the carbohydrate moiety of these proteins. The clinical manifestations are similar in all patients with sensorimotor polyneuropathy, with frequent sensory dominance and demyelinization. Atypical symptoms including motor dominance, paralysis of the cranial nerves, asymmetric forms, are rare. On histological examination, there is demyelinization and monoclonal IgM deposit along the nerve fibers. Passive in vivo and in vitro transfer experiences suggest that these monoclonal antibodies can play a role in triggering or maintaining the peripheral neuropathy. Despite identical or nearly identical antibody specificities of the monoclonal IgMs, the regions of the heavy or light chains involved vary from one patient to another, indicating that the glucoronyl sulfate determinant is quite variable. This variability is not found in other monoclonal autoantibody models with autoantibody activity such as the rheumatoid factor or cold agglutinins. These data would suggest the need for different therapeutic approaches using chemotherapy, plasmapheresis, high dose intravenous immunoglobulins and other immunomodulator treatments. Two therapeutic trials are discussed.

Antibodies, Monoclonal↗
PubMed1983

Evaluation of tissue preparation methods and paired immunofluorescence staining for immunocytochemistry of lymphomas.

Eight cross-linking fixatives were tested for preservation of extracellular or intracellular IgG, IgA, IgM, IgD, kappa and lambda light chains, J chain and secretory component. Most of the selected fixatives have been used in recent immunohistochemical studies of lymphoproliferative processes and comprised routine formalin, glutaraldehyde(1%)-formalin, Baker's formalin-calcium, formalin-sublimate, acetic acid(2%)-formalin-saline, Bouin's fluid, Susa fixative, and carbodiimide. The results obtained in artificial test substrates with defined amounts of IgG or IgA and in biological substrates (colon mucosa, tonsils, and different types of lymphomas) were compared by immunofluorescence with the antigenic preservation afforded by fixation in cold 96% ethanol (with or without inclusion of a pre-fixation 48 h washing period). An antigen concentration at least an eight-fold higher was necessary for detection with most other fixatives. Bouin's and Susa fixatives were peculiar in that they required antigen concentration 150 times higher for detection of IgG but only 3-8 times higher for IgA. Light chains were relatively well preserved by all fixatives except glutaraldehyde. For all cross-linking fixatives, the extent of antigenic masking depended on the concentration of environmental proteins, and the efficiency of unmasking with pronase or trypsin, therefore, varied with the location in the tissue. The J chain was particularly vulnerable to degradation during proteolytic treatment. The extensive masking of extracellular immunoglobulin in formalin-fixed tissue afforded a relatively good signal-to-noise ratio for immunoglobulin-producing cells when kappa and lambda chains were traced. Thus, differentiation between polyclonal and monoclonal B-cell processes on the basis of cytoplasmic labelling was often better in undigested sections. However, the light-chain type of membrane immunoglobulin could usually not be determined in directly fixed tissue. Ethanol fixation preceded by washing in saline afforded such determination and also preserved certain T-cell and HLA-DR antigens as well as diffuse alpha-naphthylbutyrate esterase. Reactive and malignant macrophages could further be traced by their cytoplasmic expression of L1 antigen, both in formalin- and ethanol-fixed material.

Antibodies↗
PubMed1982

Glycosphingolipid receptors for anti-Gd and anti-p cold agglutinins.

Anti-Gd and anti-p cold agglutinins exhibit similar serological properties: neuraminidase treatment of erythrocytes greatly reduces their agglutinability by these antibodies and protease treatment enhances their agglutination. We reported previously that an anti-p cold agglutinin was inhibited by sialosyllactoneotetraosylceramide, NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer, the most abundant ganglioside of human erythrocytes. We now report that two less abundant gangliosides are more potent inhibitors of this antibody, and of the anti-Gd antibodies, than sialosyllactoneotetraosylceramide. These two gangliosides have the same carbohydrate chain, NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc(SNH), but they differ in their ceramide moiety. The principal fatty acid of SNH-1 is C16:0, whereas SNH-2 contains a predominance of C22:0, C24:0 and C24:1. No inhibition was produced by the ganglioside, NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer. Another monoclonal cold agglutinin, Sa, which shares some serological properties with anti-Gd cold agglutinins, was not inhibited by any of these gangliosides.

Agglutinins↗
PubMed1994

Fibrinolysis during cardiac surgery. Release of tissue plasminogen activator in arterial and coronary sinus blood.

Endothelial release of tissue plasminogen activator (t-PA) may initiate fibrinolysis. Fibrinolysis and coagulation were investigated in 12 patients undergoing elective coronary artery bypass surgery. Cardiopulmonary bypass (CPB) was 108 +/- 7 min (mean +/- SEM), the time of cold, crystalloid, retrograde cardioplegia 53 +/- 5 min. Arterial and coronary sinus blood were sampled concomitantly before cardioplegia and after release of the aortic cross-clamp, for measurement of t-PA antigen (Ag) and activity, plasminogen activator inhibitor (PAI-1) Ag and activity, t-PA/PAI-1 complex, single chain urokinase (sc-uPA) and urokinase (uPA) plasminogen activators, the fibrin split product D-dimer, thrombin-antithrombin complex (TAT), and the prothrombin split product F 1 + 2. Cardiopulmonary bypass significantly increased t-PA Ag and activity, t-PA/PAI complex, D-dimer, TAT, and F 1 + 2, and decreased PAI-1 Ag and activity in arterial blood; uPA and sc-uPA were unchanged. The tissue plasminogen activator antigen was higher in coronary sinus than arterial blood after 1 (39 +/- 5 vs 24 +/- 4 ng/ml, P < 0.003), 4 (P < 0.003), and 10 min (P < 0.004) reperfusion. Tissue plasminogen activator activity and t-PA/PAI complex increased, PAI-1 activity decreased, while all other parameters were unchanged across the coronary circulation. In conclusion, CPB induces fibrinolysis and coagulation. Cold cardioplegia induces t-PA release in the coronary circulation, denoting a postischemic antithrombotic function of the coronary endothelium. Tissue plasminogen activator may be used to evaluate endothelial stimulation or injury induced by CPB, or by different regimens of myocardial protection.

Aged↗