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PubMed1975

The cold-insoluble globulin of human plasma: studies of its essential structural features.

These investigations were directed at furnishing information on the essential structural features of the cold-insoluble globulin of human plasma. Amino acid and carbohydrate analyses showed that it is a glycoprotein (1.2% sialic acid, 1.8% hexose, 2.1% hexosamine) containing all of the amino acids usually found in proteins. Circular dichroic spectral analysis suggested that cold-insoluble globulin contained a very high proportion of beta-structure; no evidence for the presence of alpha-helix was found. Sedimentation velocity experiments at pH 7.0, in the presence or absence of dithiothreitol, plus related gel electrophoretic experiments at pH 8.4, indicated that the integrity of certain disulfide bridges was necessary for its solubility under "physiologic" conditions. In experiments in urea-containing solution, two sedimenting peaks were observed. The major one, amounting to more than 95% of the total, had an s20,w of 5.6 S, the minor peak had an s20,w of 7.3 S. Following disulfide bridge reduction a single symmetrical peak of 3.9 S was formed. Such behavior suggested that cold-insoluble globulin is a multichain molecule whose subunit chains are linked by disulfide bridging. Strong support for this conclusion was obtained from electrophoretic analyses in gels containing dodecylsulfate, in that cold-insoluble globulin manifested an increased rate of migration after reduction of disulfide bridges. The reduced cold-insoluble globulin band could be resolved into a closely spaced doublet, the components of which had molecular weights of 220 000 and 215 000, respectively. Since in sedimentation equilibrium experiments the molecular weight of the unreduced molecule was estimated to be 450 000, values in this range for the size of the subunit chain suggested that each cold-insoluble globulin molecule is composed of two covalently linked chains. The nature of the size heterogeneity of the reduced subunit chains is uncertain. However, the finding of a single type of NH2-terminal sequence ([Glu-Ala) in cold-insoluble globulin preparations, is consistent with the speculation that the smaller subunit may be a catabolic intermediate arising via release of peptide material containing the COOH-terminus of a parent chain.

Amino Acids↗
PubMed1993

Resistance to ischaemic nerve-fibre block in diabetes mellitus and in mitochondrial encephalomyopathies.

Thermal thresholds were measured during ischaemic compression block in the left forearms of 26 healthy subjects, 10 patients with diabetes mellitus and 6 patients suffering from different kinds of mitochondrial disorders. Cold and warm thresholds in the 6 patients with deficiencies in the respiratory chain increased earlier than in normals. When cold perception was impaired, cold stimuli were perceived as warmth and pinprick perception attenuated. In diabetics cold thresholds were less elevated during ischaemic block than in controls. This was paralleled by tingling paraesthesiae in all groups. The findings show that higher resistance to ischaemic nerve-fibre block in diabetes mellitus is not exclusively based on increased anaerobic metabolism.

Adult↗
PubMed1982

Reactivity of cold agglutinins with subsets of human lymphocytes of various origins.

Forty-five cold agglutinins (CA) were tested against various populations of lymphocytes by cytotoxicity and immunofluorescence assays. Marked differences were observed between anti-I and anti-i CA. Thirty-six per cent of anti-I killed preferentially peripheral blood and tonsillary B lymphocytes, whereas only 12 per cent killed preferentially T lymphocytes. Anti-I killed a much higher proportion of B-chronic lymphocytic leukemia cells than peripheral blood lymphocytes, peripheral blood B cells or T-chronic lymphocytic leukemia cells. Forty-three per cent of anti-i killed preferentially peripheral blood T lymphocytes and 54 per cent killed preferentially tonsillary T cells, whereas only 14 per cent killed more peripheral blood B cells and none killed preferentially tonsillary B cells. The kill of thymic lymphocytes and T-chronic lymphocytic leukemia cells by anti-i was very high, whereas the kill of B-chronic lymphocytic leukemia cells was very low. Almost all CA of other than I-i specificities showed preferential kill of peripheral blood B (83 per cent) and tonsillary B (67 per cent) cells. Tonsillary lymphocytes were usually more susceptible to the cytotoxic activity of CA than peripheral blood lymphocytes. Cold agglutinins with kappa light chains killed more B cells whereas CA with gamma light chains seemed to kill more T cells. Cytotoxicity did not correlate to the utilization of complement. It is suggested that the density and/or the accessibility of membranous antigens may be different on B and T cells, or alternatively that in addition to antigens common to all lymphocytes, anti-I and non-I/i cold agglutinins recognize specific antigenic determinants on B lymphocytes, whereas anti-i cold agglutinins recognize specific antigenic determinants on T lymphocytes.

Agglutinins↗
PubMed1996

Lack of effect of recombinant human superoxide dismutase on cold ischemia-induced arteriosclerosis in syngeneic rat aortic transplants.

Prolonged cold ischemia time and the generation of free oxygen radicals during reperfusion are risk factors for allograft arteriosclerosis. Growth factors are the main pro-proliferative mediators of smooth muscle cells in classical and in allograft arteriosclerosis. Superoxide dismutase is an enzyme that catalyzes the dismutation of superoxide anions into hydrogen peroxide. This study was designed to investigate which smooth muscle cell growth factor contribute to the formation of arteriosclerosis in syngenic vascular grafts with prolonged ischemia time, and whether perioperative intravenous administration of recombinant human superoxide dismutase (rh-SOD) prevents arteriosclerosis in these grafts. DA aortas were transplanted into DA recipients. One group of transplants was made with a short ex vivo ischemia time (15 min), while the other group transplant grafts was stored for 24 hr in cold saline. In addition to morphometric quantitation of the histological alterations, RNA isolated from grafts with short cold ischemia time in a semiquantitative polymerase chain reaction specific for various known smooth muscle cell growth factors. Syngeneic grafts with prolonged cold ischemia time showed severe intimal thickening and prominent medical necrosis, which were not seen in control groups. Approximately 3-fold levels of insulin-like growth factor-1 were found in ischemic syngeneic grafts compared with non-ischemic syngenic grafts, whereas epidermal growth factor levels were slightly lower. No changes in other growth factor mRNAs were found. Perioperative treatment with rh-SOD did not have significant effect on the extent of intimal thickening nor on the intensity of medial necrosis in grafts with prolonged ischemia time, and administration of rh-SOD did not change the expression level of insulin-like growth factor-1 in the grafts, either.

Animals↗
PubMed2001

Cold single-strand conformation polymorphism analysis: optimization for detection of APC gene mutations in patients with familial adenomatous polyposis.

Over 200 adenomatous polyposis coli (APC) gene mutations have been described in familial adenomatous polyposis (FAP) patients. Recent single-strand conformation polymorphism (SSCP) screening methods have introduced minigel runs, simple ethidium bromide staining and external temperature control without any loss of sensitivity (cold-SSCP). In order to test the effectiveness in APC mutation detection, cold-SSCP was employed following polymerase chain reaction (PCR) amplification in three patients with FAP. Different running parameter combinations were compared. The three mutations already known were all diagnosed by cold-SSCP. The gel concentration was found to be essential in detecting the single-base substitution in fragments of different lengths. The observation of deletions was not affected by gel concentrations and heteroduplex bands were always produced. The temperature or glycerol addition did not significantly affect sensitivity. This modified cold-SSCP method provides a simple and effective way for detecting several known Apc gene mutations without any loss of sensitivity and could be useful for large-scale molecular diagnosis of FAP.

Adenomatous Polyposis Coli↗
PubMed2001

Cold-induced increment in rat adrenal gland type II deiodinase is corticosterone dependent.

In this study we analyzed whether corticosterone synthesis is involved in the regulation of adrenal gland type II deiodinase (AG-D2) activity during acute cold exposure. Two well-known inhibitors of steroidogenesis, aminoglutethimide (AGT) and metyrapone (MTP), were administered to male Wistar rats maintained either at room temperature or acutely exposed to cold (1 h at 4 degrees C). AG-D2 activity was measured by the radioiodide release method, and corticosterone circulating levels were measured by competitive protein binding assay. Results show that resting corticosterone levels and AG-D2 activity were lower in both AGT- and MTP-treated rats. Furthermore, the phasic increase normally exhibited by AG-D2 activity in response to acute cold stress was blunted in AGT- and MTP-treated animals. Therefore, we conclude that corticosterone synthesis is necessary in preserving the physiologic response of AG-D2 activity to cold exposure.

Adrenal Glands↗
PubMed2004

Quantitative and qualitative analysis of rhinovirus infection in bronchial tissues.

Although rhinovirus (RV) infections can cause asthma exacerbations and alter lower airway inflammation and physiology, it is unclear how important bronchial infection is to these processes. To study the kinetics, location, and frequency of RV appearance in lower airway tissues during an acute infection, immunohistochemistry and quantitative polymerase chain reaction analysis were used to analyze the presence of virus in cells from nasal lavage, sputum, bronchoalveolar lavage, bronchial brushings, and biopsy specimens from 19 subjects with an experimental RV serotype 16 (RV16) cold. RV was detected by polymerase chain reaction analysis on cells from nasal lavage and induced sputum samples from all subjects after RV16 inoculation, as well as in 5 of 19 bronchoalveolar lavage cell samples and in 5 of 18 bronchial biopsy specimens taken 4 days after virus inoculation. Immunohistochemistry detected RV16 in 39 and 36% of all biopsy and brushing samples taken 4 and 15 days, respectively, after inoculation. Infected cells were primarily distributed in discrete patches on the epithelium. These results confirm that infection of lower airway tissues is a frequent finding during a cold and further demonstrate a patchy distribution of infected cells, a pattern similar to that reported in upper airway tissues.

Adult↗
PubMed2004

Effects of 20-week intermittent cold-water-immersion on phenotype and myonuclei in single fibers of rat hindlimb muscles.

The effects of 20 weeks of intermittent cold-water-immersion on myosin heavy chain (MHC) expression,cross-sectional area (CSA), myonuclear number, and myonuclear domain size in isolated single fiber of soleus and extensor digitorum longus (EDL) muscles were studied in male Wistar rats. Cold exposure was accomplished by submerging the rats in shoulder-deep water, maintained at approximately 18 degrees C, for 1 hour/day, 5 days/week and for 20 weeks. Cold exposure resulted in a significant inhibition of body and soleus muscle weight gain. The percent type IIa MHC fibers of EDL muscle was increased, whereas that of type IIa + b MHC fibers was less in cold-exposed group than controls (p < 0.05). The mean CSA and myonuclear number in type I MHC fibers of soleus muscle in cold-exposed group were significantly less than controls. Myonuclear domain in type IIa fibers of EDL in the cold-exposed group was greater than controls (p < 0.05). It is suggested that prolonged cold exposure causes the fiber-type-specific adaptation in rat hindlimb muscles. It is further indicated that cold-exposure-related modulation of myonuclear number was closely related to reduction of fiber CSA, not the shift of fiber phenotype.

Animals↗
PubMed1998

Human monoclonal antibodies encoded by the V4-34 gene segment show cold agglutinin activity and variable multireactivity which correlates with the predicted charge of the heavy-chain variable region.

We have characterized the reactivities of a panel of V4-34-encoded human IgM monoclonal antibodies (mAb) which bind the erythrocyte Rh D antigen, derived from an immunized individual. These were compared with the specificities of V4-34-encoded autoantibodies with I/i reactivity produced from patients with cold agglutinin disease (CAD), and other V4-34-encoded autoantibodies. The antibodies were evaluated for cold agglutinin activity using haemagglutination tests, immunofluorescence microscopy for reactivity with tissue components, and in solid phase radiobinding assays with purified antigens. We found that (i) cold agglutinin activity was a property of all the V4-34-encoded mAb (ii) the cold agglutinins from CAD patients were generally monospecific for I/i whereas most of the anti-D and the other V4-34-encoded mAb displayed multireactive properties, frequently binding to strongly acidic antigens (iii) computation of the net charge of the heavy-chain V regions showed that the multireactive mAb were generally more positively charged than the monospecific cold agglutinins, which could contribute to their multireactive phenotype. The involvement of charge interactions was further indicated by the effects of pH and ionic strength on the immunofluorescence staining patterns.

Adult↗
PubMed2003

Regulation of human immune gene expression as influenced by a commercial blended Echinacea product: preliminary studies.

Consumption of Echinacea at the first sign of symptoms has been clinically shown to reduce both the severity and duration of cold and flu. Quantitative polymerase chain reaction optimized for precision and reproducibility was utilized to explore in vitro and in vivo changes in the expression of immunomodulatory genes in response to Echinacea. In vitro exposure of THP-1 cells to 250 microg/ml of Echinacea species extracts induced expression (up to 10-fold) of the interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, intracellular adhesion molecule, interleukin-8, and interleukin-10 genes. This preliminary result is consistent with a general immune response and activation of the nonspecific immune response cytokines. In vivo gene expression within peripheral leukocytes was evaluated in six healthy nonsmoking subjects (18-65 years of age). Blood samples were obtained at baseline and on Days 2, 3, 5, and 12 after consuming a commercial blended Echinacea product, three tablets three times daily (1518 mg/day) for two days plus one additional dose (506 mg) on day three. Serum chemistry and hematological values were not different from baseline, suggesting that liver or bone marrow responses were not involved in acute responses to Echinacea. The overall gene expression pattern at 48 hr to 12 days after taking Echinacea was consistent with an antiinflammatory response. The expression of interleukin-1beta, tumor necrosis factor-alpha, intracellular adhesion molecule, and interleukin-8 was modestly decreased up through Day 5, returning to baseline by day 12. The expression of interferon-alpha steadily rose through Day 12, consistent with an antiviral response. These preliminary data present a gene expression response pattern that is consistent with Echinacea's reported ability to reduce both the duration and intensity of cold and flu symptoms.

Adolescent↗
PubMed1988

Histochemical heterogeneity of intrafusal muscle fibres in slow and fast skeletal muscles of the rat.

Intrafusal muscle fibres of the slow soleus (Sol) and fast vastus lateralis (VL) muscles of the rat were studied histochemically. Serial transverse sections were incubated for the localization of succinate dehydrogenase (SDH), alpha glycerophosphate dehydrogenase (GPD) and adenosine triphosphatase (ATPase). The latter was examined further after preincubation in acidic solution held at either low or room temperature (RT). The bag2 intrafusal fibres in both muscles displayed high regular and acid stable ATPase, but low SHD and GPD activities. Bag1 intrafusal fibres showed low to moderate regular ATPase, a regional heterogeneity after RT acid preincubation (low activity in juxtaequatorial and high in polar zones), moderate SDH, but low GPD reactions. In both muscles the chain fibres usually exhibited high ATPase for both regular and cold acid preincubated reactions, but usually low activity after RT acid preincubation; they had high SDH but variable GPD activities. In Sol muscle, however, approximately 25% of spindles contained chain fibres that showed high acid-stable ATPase reaction after both cold and RT acid preincubation. In contrast, chain fibres in some VL spindles had a characteristically low ATPase reaction even after cold acid preincubation. This study, therefore, has delineated the existence of an inherent heterogeneity among chain fibres (with respect to their histochemical reactions) in muscle spindles located within slow and fast muscles and also between those found within populations of either Sol or VL muscle spindles.

Animals↗
PubMedDate not supplied

Cold agglutinin activity is common among human monoclonal IgM Rh system antibodies using the V4-34 heavy chain variable gene segment.

BACKGROUND: The V4-34 gene segment is commonly used by human monoclonal IgM alloantibodies against blood group antigens and by cold-reactive red cell autoantibodies with anti-I or anti-i specificity. This study was conducted to determine whether cold agglutinin activity is found among the V4-34-encoded alloantibodies. STUDY DESIGN AND METHODS: Fifty-four human IgM monoclonal antibodies (MoAbs) against Rh system antigens were tested for cold agglutinin activity against red cells lacking the relevant Rh system antigen and for reactivity with tissue I and/or i antigens using immunohistochemistry. The findings were correlated with the utilization of the V4-34 segment as determined in an enzyme-linked immunosorbent assay with an antibody (9G4) that is specific for this gene product and were also correlated with other serologic properties. RESULTS: Of the MoAbs, 59 percent were 9G4-positive. Of the 9G4-positive subset, 16 and 44 percent agglutinated native adult (express I) and cord (express i) cells, respectively, at 4 degrees C; these levels rose to 84 and 94 percent, respectively, with the use of papain-treated cells. The red cell antigens recognized at 4 degrees C were cleaved by endo-beta-galactosidase, which is consistent with their being I and i. Of the 9G4-positive subset, 53 percent bound to tissue i antigen. These reactivities were not found among 9G4-negative MoAbs. Endo-beta-galactosidase treatment of red cells enhanced Rh system antibody agglutination by 9G4-negative MoAbs. CONCLUSION: Anti-I/i reactivity is common among IgM Rh system MoAbs and is shown only by the V4-34-encoded subset. This finding has implications for the use of MoAbs for Rh system typing of blood.

Agglutinins↗
PubMed2005

Mutations in the "lid" region affect chain length specificity and thermostability of a Pseudomonas fragi lipase.

The cold-adapted Pseudomonas fragi lipase (PFL) displays highest activity on short-chain triglyceride substrates and is rapidly inactivated at moderate temperature. Sequence and structure comparison with homologous lipases endowed with different substrate specificity and stability, pointed to three polar residues in the lid region, that were replaced with the amino acids conserved at equivalent positions in the reference lipases. Substitutions at residues T137 and T138 modified the lipase chain-length preference profile, increasing the relative activity towards C8 substrates. Moreover, mutations conferred to PFL higher temperature stability. On the other hand, replacement of the serine at position 141 by glycine destabilized the protein.

Amino Acid Sequence↗
PubMed1989

A myosin isoform repressed in hypertrophied ALD muscle of the chicken reappears during regeneration following cold injury.

A library of monoclonal antibodies specific for myosin heavy chain (HC) was used to study myosin expression in regenerating fibers. The response to cold injury of slow skeletal ALD muscle previously induced to eliminate SM1 myosin by weight overload was compared to that of its contralateral control. Native gel electrophoresis combined with immunoblotting demonstrated that slow SM1 myosin HC eliminated from hypertrophic muscle reappeared both at the site of active regeneration and unexpectedly, also distal to the site of injury. The regeneration response of hypertrophied muscles was similar to that of the controls. In addition to SM1 myosin HC, ventricular-like and embryonic/fast isoforms were also expressed in both muscles during the early stages of regeneration and disappeared as the muscle fibers matured. These observations demonstrate that regenerating slow muscle fibers reexpress myosins' characteristic of developing muscle irrespective of the myosin phenotype prior to injury. The reappearance of repressed myosin HC in the hypertrophied ALD muscle is consistent with the presence of newly differentiated myonuclei.

Animals↗
PubMed1980

Fatty acid patterns during plasma fractionation.

When albumin is prepared from human blood plasma by the cold ethanol method, nonesterified long chain fatty acids present in the plasma do not strictly copurify with albumin. About half of them are lost to the varioius globulin fractions precipitated in the first steps of the fractionation procedure. A different behavior is observed for the short chain fatty acid, caprylic acid. On the other hand, if fractionation of plasma proteins is done by ammonium sulfate precipitation, both long and short chain fatty acids remain with the albumin.

Ammonium Sulfate↗
PubMed2001

Identification of model structure for aquatic ecosystems using regionalized sensitivity analysis.

The Regionalized Sensitivity Analysis (RSA) was developed in 1978, for identifying critical unknown processes in poorly defined systems, thus directing the focus of further scientific investigations. Here, we demonstrate its application to model structure identification, by ranking the constituent hypotheses and identifying the critical elements for progressive revision of the model. Our case study is Lake Oglethorpe--a small monomictic impoundment in South-eastern Georgia, USA. Recent studies indicate that the warm temperate regional climate affords an extended growing season--typically from March to October--which promotes bacterial productivity in the lake. The result is a summer food web dominated by microbial processes, in contrast to the conventional phytoplankton-dominated food chains typically observed in the cold temperate lakes of Europe and North America. Starting with a simple phytoplankton-based food web model and a qualitative definition of system behaviour, we use the RSA procedure to establish the critical role of bacteria-mediated decomposition in Lake Oglethorpe, thus justifying the inclusion of microbial processes. Further analysis reveals the importance of size-dependent selective consumption of phytoplankton and bacteria. Finally, we discuss important practical implications of this novel application of the RSA regarding sampling efficiency and statistical robustness.

Animals↗
PubMedDate not supplied

[Hydration of the left spiral of the poly-L-proline type. Study by the Monte Carlo method].

The paper exhibits results of hydration shell Monte Carlo calculations in poly-L-proline II and extended helix conformation and in alpha-helical and beta-structural conformations for comparison. It was found that left-handed helix of poly-L-proline II type as well as epsilon-helix are characterized by very favorable hydration. Therefore this conformation has preference as compared to other standard conformations of the main polypeptide chain. This determined inevitability of cold denaturation of protein.

Amino Acid Sequence↗