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PubMed1993

Beta 1 and beta 4 integrin expression in methacarn and formalin-fixed material from in situ ductal carcinoma of the breast.

The integrins are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Previous distribution studies on integrin expression have been limited by the requirement of cryostat sectioned tissues, and consequent poor resolution. We have examined 40 examples of ductal carcinoma in situ (DCIS) for the expression of both beta 1 and beta 4 integrin chains. These showed strong polarized membrane staining of residual myoepithelial cells (correlating with expression of smooth muscle specific actin) and of the basement membrane region with beta 1 and beta 4 antibodies respectively. In 12 out of 40 cases, the DCIS was negative for the beta 1 chain and a variable pattern of reactivity was seen in the remaining cases. The beta 4 chain was detected focally and weakly in the tumour cells of 7/40 DCIS and strongly in one; all of these cases were also positive for the beta 1 chain. Of the 22 cases where co-existent invasion was present, the infiltrating component showed either a similar degree or a diminution of the extent of immunostaining when compared with the in situ component; only one showed enhanced staining (beta 1 only). This study demonstrates that two of the main beta chains, beta 1 and beta 4, can be effectively demonstrated on methacarn and cold (4 degrees C) formalin-fixed tissues by avidin-biotin indirect immunoperoxidase staining and that the results are similar to those achieved using frozen tissue.

Acetates↗
PubMed1979

A histoenzymatic study of rat intrafusal muscle fibres.

The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (alpha-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K2-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkali-preincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K2-EDTA preincubation. Following either acetic acid solution or cold and room temperature K2-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K2-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing alpha-GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles.

Adenosine Triphosphatases↗
PubMed2004

The alternative respiratory pathway of euglena mitochondria.

Mitochondria, isolated from heterotrophic Euglena gracilis , have cyanide-resistant alternative oxidase (AOX) in their respiratory chain. Cells cultured under a variety of oxidative stress conditions (exposure to cyanide, cold, or H2O2) increased the AOX capacity in mitochondria and cells, although it was significant only under cold stress; AOX sensitivity to inhibitors was also increased by cold and cyanide stress. The value of AOX maximal activity reached 50% of total respiration below 20 degrees C, whereas AOX full activity was only 10-30% of total respiration above 20 degrees C. The optimum pH for AOX activity was 6.5 and for the cytochrome pathway was 7.3. GMP, AMP, pyruvate, or DTT did not alter AOX activity. The reduction level of the quinone pool was higher in mitochondria from cold-stressed than from control cells; furthermore, the content of reduced glutathione was lower in cold-stressed cells. Growth in the presence of an AOX inhibitor was not affected in control cells, whereas in cold-stressed cells, growth was diminished by 50%. Cyanide diminished growth in control cells by 50%, but in cold-stressed cells this inhibitor was ineffective. The data suggest that AOX activity is part of the cellular response to oxidative stress in Euglena .

Animals↗
PubMed2001

Engineering the properties of a cold active enzyme through rational redesign of the active site.

In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.

Alkaline Phosphatase↗
PubMedDate not supplied

Characterization of various anti-Pr cold agglutinins.

Forty -one samples of anti-Pr cold agglutinins (CA) were studied. The titers ranged from 4 to 32,000. As is the case with cold agglutinins of other specificities, immunoglobulin class M and k-type light chains predominated with anti-Pr CA. On the other hand, the few IgM lambda, IgG, and IgA found were associated preferentially with anti-Pr specificity. All anti-Pr CA were inhibited by human red cell membrane sialoglycoproteins. On the basis of an increased inhibition by sialoglycoproteins after periodate oxidation and carbodiimide treatment, respectively, three anti- Pr2 and five anti-Pr3 were found. Among 24 anti-Pr1 CA subclassified on the basis of agglutination with dog red cells, six were anti- Pr1h , 16 were anti- Pr1d , and two did not fit into the subclassification. Similar to the anti- Pr1h , - Pr1d subclassification, anti-Pr3 CA could be subclassified into anti- Pr3h , - Pr3d . Three anti-Pra examples were found. None of the anti-Pr CA was inhibited by N-acetylneuraminic acid; some (5/35) were inhibited by sialyllactose, NeuAc alpha 2-3 2-6 Gal beta 1-4Glc, in high doses (5.0-20.0 mM).

Aged↗
PubMed1978

[Increase in heat production by muscle contraction caused by 2,4-dinitrophenol].

By means of a highly sensitive thermometric method (1.10--50 C/mm) it was possible to demonstrate that the heat production during a single contraction of the isolated rat diaphragm increased under the influence of 2,4-dinitrophenol (DNP). Low DNP concentrations failed to produce any manifest changes in the resting muscle, but its action was distinct during contraction. With the same muscle contraction force DNP action was followed by an increase (about 1 1/2-fold) of heat production. This fact confirmed a supposition on the leading role of oxidation and phosphorylation uncoupling in the muscle cell respiratory chain in the increase of muscle contraction heat production in the animals after cold adaptation.

Animals↗
PubMed1982

Tumor immunity to murine plasma cell tumors. VII. Expression of H-2 and tumor antigens on Ig synthesis variants of MPC-11.

As a part of our continuing investigations directed toward defining the nature of the antigens on murine plasmacytomas (PCT), MPC-11 and two immunoglobulin (Ig) synthesis variants of this cell line were examined for H-2 and tumor-associated antigen (TAA) expression, and for the possible role of H-2 in the cytotoxic T lymphocyte response to TAA. The tumor lines studied were MPC-11 (an IgG2b producer), MPC-11,662 (a variant which produces only light chains), and NP.2 (a variant which produces neither Ig chain). Both H-2 expression, which was analyzed by flow cytometry and by cold target cell blocking of the activity of cytotoxic T lymphocytes (CTL) generated in allogeneic cultures, and TAA expression, which was analyzed by cold target cell blocking of the activity of tumor-specific CTL generated in syngeneic cultures, were found to be quantitatively similar on the three lines. When a possible association between H-2 and TAA expression was examined by testing the ability of anti H-2 sera to block tumor-specific CTL directed against MPC-11, no association between H-2 and TAA was detected. The results of these studies provide new information about H-2 and TAA expression on murine PCT. Furthermore, they suggest that changes in H-2 and TAA expression which have been reported to sometimes accompany loss of Ig synthesis may represent independent genetic, phenotypic, or metabolic variations, rather than alterations linked to the loss of Ig synthesis. In addition, these results, in agreement with out previous studies on MPC-11, support the concept that there is not an obligatory association of TAA with H-2 on the surface of murine PCT. Because these results differ from those reported in some other systems in which an association between H-2 and TAA on murine PCT was demonstrated, it must be emphasized that the TAA of PCT appear to be quite heterogeneous, and more investigations will be necessary in order to clearly define these antigen and the T-cell response to each of them.

Animals↗
PubMedDate not supplied

[The characteristics of the expression of temperature-dependent dominant lethal mutations in Drosophila melanogaster].

The results of a series of investigations on obtaining and analysing dominant temperature-sensitive lethals (DTS-lethals) are summarized. Using EMS, both cold- and heat sensitive mutations of this type were induced in large autosomes. The cold-sensitive mutations of chromosome 3 were revealed for the first time. The effect of genetic phone on DTS lethal penetrance and expressivity was studied. The character of expression of two mutations possessing a pleiotropic action was examined. The heat-sensitive 1(2)M90DTS mutation disturbed the structure of abdominal tergites in all imago and, besides, caused an abrupt decrease in the female reproductive period. The pattern of alterations observed in oogenesis along with the result of phenogenetic analysis suggest that this gene takes part in the genetic control over the proliferation of stem oogonial cells. The cold-sensitive 1(2)M66DCS mutation caused a number of disturbances in imago thoracic structures: inability to flight, raised up wings, extremity fracture, etc. Mutant flies showed serious disorders of indirect flight muscles both at morphological and at ultrastructural levels. It was established that this mutation was a cold-sensitive allele of Mhc gene which controls the synthesis of myosine heavy chains in drosophila.

Alleles↗
PubMed1997

Pyrophosphorylases in potato. V. Allelic polymorphism of UDP-glucose pyrophosphorylase in potato cultivars and its association with tuber resistance to sweetening in the cold.

UDP-glucose pyrophosphorylase (UGPase) was cloned from six American and nine European potato (Solanum tuberosum L.) cultivars. Restriction mapping of the different UGPase-cDNAs with BamHI, HindIII, and EcoRI revealed that at least two mRNA populations were present in most cultivars. Staining for UGPase activity in nondenaturing gels of proteins extracted from developing potato tubers yielded two major isozymes that were highly active and appeared to be dimeric in nature. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all isozymes were disassociated into a single subunit with a molecular mass of 53 kD. Since UGPase has been demonstrated to be a single-copy gene in the haploid genome of potato (A.Y. Borovkov, P.E. McClean, J.R. Sowokinos, S.H. Ruud, G.A. Secor [1995] J Plant Physiol 147: 644-652), there must be allelic differences at the UGPase locus (chromosome 11). The two alleles, designated ugpA and ugpB, were identified by the absence and presence of a BamHI site, respectively. The relative band intensities of the two cDNA populations following polymerase chain reaction amplification and agarose gel electrophoresis were related to a potato cultivar's ability to resist sweetening when exposed to cold temperatures.

Alleles↗
PubMed1993

Molecular characterization of a cross-reactive idiotope on human immunoglobulins utilizing the VH4-21 gene segment.

The anti-idiotypic (anti-Id) antibody (Ab) 9G4 binds a cross-reactive idiotope (CRI) present in a select group of human autoantibodies. This Id has been localized to the portion of immunoglobulin (Ig) heavy (H) chains encoded by the VH4-21 gene segment, a member of the human VH4 family. This gene segment is utilized by essentially all cold agglutinin (CA) Abs with I/i specificity isolated from patients with CA disease stemming from chronic lymphoproliferative disorders. In this study, mutational analysis of a CA has been used to determine the structural basis for 9G4 binding to Abs utilizing the VH4-21 gene segment. Recombinant CA H chain mutants were produced and their 9G4 reactivity determined. Mutants were generated by exchanging VH4-21 sequences in the FR1, CDR1, and CDR2 with corresponding sequences from a closely related gene segment V71-2, a VH4 family member that is associated neither with Abs having CA activity nor with Abs that react with 9G4. The results indicate that the motif AVY at amino acid positions 23-25 in FR1 defines the 9G4 idiotope. Reaction of these recombinant Abs with a polyclonal rabbit anti-CA antiserum absorbed to render it specific for a CA CRI also maps predominantly to FR1. These findings indicate that the solvent-exposed FR1 plays an important role in eliciting an immune response to Igs.

Agglutinins↗
PubMed2004

Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva.

BACKGROUND: Saliva is a potentially useful source of genomic DNA for genetic studies since it can be collected in a painless and non-invasive manner. We sought to determine whether different storage conditions of saliva samples impact our ability to extract genomic DNA that is of sufficient quality for use in the polymerase chain reaction (PCR). METHODS: Saliva was collected from healthy volunteers and 2-ml aliquots subjected to different storage conditions: S1--washing of saliva using phosphate-buffered saline (PBS) and extraction of DNA on the same day of collection; S2--washing and centrifugation to yield a pellet, which was stored at-70 degrees C for 1 week prior to DNA extraction; S3--storage of whole saliva at 4 degrees C for 7 days, followed by washing and extraction of DNA; S4--storage at 4 degrees C for 7 days, followed by washing and pellet formation. The pellet was stored at -70 degrees C for 1 month before extraction of the DNA; S5--storage at-70 degrees C for 1 month, followed by washing and extraction of DNA. DNA yield and purity was determined by spectrophotometry at 260 and 280 nm. Twenty nanograms of genomic DNA was used for the polymerase chain reaction, and the resulting PCR band was captured by digital photography and quantified. RESULTS: The amounts of DNA extracted from 2 ml of saliva varied widely under the different storage conditions, while purity of the DNA extraction, based on OD(260/280) ratios, was good and comparable. PCR resulted in the presence of a single specific product of the correct size from all samples regardless of saliva storage conditions. Quantification of PCR bands showed significant differences between the various storage conditions (P<0.05). Compared to S1 samples, PCR bands from conditions S2 and S3 were not as strong, while those amplified from S4 and S5 samples were the weakest. Post-hoc analyses showed that the means for conditions S4 and S5 were significantly different from S1-S3. Qualitatively similar results were obtained when the PCR experiment was repeated. CONCLUSIONS: Saliva can act as a useful source of genomic DNA, even when stored under less than optimal conditions.

Cold Temperature↗
PubMed1986

Expression of platelet glycoprotein Ib alpha in HEL cells.

We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.

Asialoglycoproteins↗
PubMed2001

Respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease.

The effects of respiratory viral infection on the time course of chronic obstructive pulmonary disease (COPD) exacerbation were examined by monitoring changes in systemic inflammatory markers in stable COPD and at exacerbation. Eighty-three patients with COPD (mean [SD] age, 66.6 [7.1] yr, FEV(1), 1.06 [0.61] L) recorded daily peak expiratory flow rate and any increases in respiratory symptoms. Nasal samples and blood were taken for respiratory virus detection by culture, polymerase chain reaction, and serology, and plasma fibrinogen and serum interleukin-6 (IL-6) were determined at stable baseline and exacerbation. Sixty-four percent of exacerbations were associated with a cold occurring up to 18 d before exacerbation. Seventy-seven viruses (39 [58.2%] rhinoviruses) were detected in 66 (39.2%) of 168 COPD exacerbations in 53 (64%) patients. Viral exacerbations were associated with frequent exacerbators, colds with increased dyspnea, a higher total symptom count at presentation, a longer median symptom recovery period of 13 d, and a tendency toward higher plasma fibrinogen and serum IL-6 levels. Non-respiratory syncytial virus (RSV) respiratory viruses were detected in 11 (16%), and RSV in 16 (23.5%), of 68 stable COPD patients, with RSV detection associated with higher inflammatory marker levels. Respiratory virus infections are associated with more severe and frequent exacerbations, and may cause chronic infection in COPD. Prevention and early treatment of viral infections may lead to a decreased exacerbation frequency and morbidity associated with COPD.

Aged↗
PubMed1996

Bronchial inflammation and the common cold: a comparison of atopic and non-atopic individuals.

BACKGROUND: Cold virus infections are associated with asthma attacks and with increased bronchial responsiveness even in normal subjects. Possible mechanisms include epithelial damage, interaction with adhesion molecules or with T-helper cell subsets. OBJECTIVE: To determine whether colds increase lower airway inflammation, comparing atopic with non-atopic normal subjects. METHODS: Thirty healthy volunteers (15 atopic) took part. Baseline tests included viral serology, microbiological culture and polymerase chain reaction for rhinovirus infection (HRV-PCR), histamine bronchial provocation and bronchoscopy. Twenty subjects (eight atopic) underwent repeat tests when they developed a cold. RESULTS: Forced expiratory volume in one second (FEV1) was significantly lower during colds (-0.19 L [95% confidence interval -0.10, -0.29], P = 0.0004) and there was a significant increase in bronchial responsiveness (+0.62 doublings of the dose-response slope [+0.24, +1.00], P = 0.003). Eight subjects (two atopic) had a diagnosed viral infection: two HRV, three coronavirus (HCV), one HRV + HCV, one parainfluenza III (PI) and one respiratory syncytial virus (RSV) (also Haemophilus influenzae). In biopsies, during colds, total eosinophils (EGI+) increased significantly (geometric mean 6.73-fold [1.12,40.46], P = 0.04). Activated eosinophils (EG2+) only increased significantly in the subgroup without diagnosed viral infection and particularly in atopic rhinitics. T-suppressor (CD8+) cells also increased significantly (median + 178.3 cells mm2, P = 0.004). Epithelial expression of intercellular adhesion molecule-1 (ICAM-1) expression increased in four atopic rhinitics during colds. Bronchial washings showed a significant increase in neutrophils (GM 1.53-fold [1.04,2.25], P = 0.02). CONCLUSION: Lower airway inflammation was present in atopic and non-atopic normal subjects with colds. Atopic subjects differed in that they were less likely to have positive virological tests and were more likely to show activated eosinophilia in the lower airway, despite a similar spectrum of symptoms.

Adolescent↗
PubMed1975

The intracellular localization of long-chain acyl-CoA synthetase in brown adipose tissue.

1. The acyl-CoA synthetase activity in brown adipose tissue of cold-exposed guinea pig has been studied by measuring the rate of palmitoylcarnitine formation in the presence of excess carnitine palmitoyltransferase. 2. The rate of palmitoylcarnitine formation in the mitochondria was found to be 161 plus or minus 64 nmol.mg-minus-1. min-minus-1 (n=9). 3. In the absence of added palmitate and bovine serum albumin a total of 35 plus or minus 1 nmol endogenous fatty acids.mg-minus-1 were activated with three different mitochondrial preparations. 4. Three different experimental approaches have been used to study the subcellular localization of the enzyme: (a) conventional differential centrifugation (De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Biochem. J. 60, 604-617) (B) the determination of the sediterm of different marker enzymes (Slinde, E. and Flatmark. T. (1973) Anal. Biochem. 56, 324-340) and (c) the determination of the stoichiometry between the activities of these enzymes sedimented at higher centrifugal effects. 5. Throughout all fractionation procedures, the long-chain acyl-CoA synthetase follows strictly the amine oxidase generally considered to be exclusively located on the mitochondrial outer membrane.

Adipose Tissue, Brown↗