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At least 343 records · Page 19Linked to original sources
PubMed2001

Alterations of K-ras and p53 mutations in colorectal cancer patients in Central Europe.

Many molecular investigations of colorectal cancer (CRC) have suggested that the accumulation of specific mutations in proto-oncogenes and tumor suppressor genes regulating cell growth via signal transduction trigger the stagewise progression to malignancy. In this study, the frequency, location, and type of mutations of the K-ras proto-oncogene exon I and p53 tumor suppressor gene exons 5-8 were analyzed in colorectal carcinomas of 65 patients from Central Europe, using polymerase chain reaction (PCR)-cold single-strand conformation polymorphism (SSCP) screening and direct sequencing. The incidence of K-ras activating mutations in these Central European samples was lower (25%) compared to that obtained in American and western European populations (40-50% at least), while the incidence of p53 inactivating mutations was similar (58%). These results suggest that some other genetically linked mechanisms may play a role in CRC development and progression, and hence K-ras and p53 mutations cannot be considered to be universal genetic markers for CRC.

Colorectal Neoplasms↗
PubMed1985

Evidence for the interaction of alpha-actinin and calmodulin with the clathrin heavy chain.

In the present study protein overlays were used to study the molecular interactions of clathrin with clathrin coat-associated proteins. Coated vesicles (CV) were isolated, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose. The transfers were quenched and equilibrated in buffer, containing 1% Triton X-100. Alpha-Actinin and calmodulin, proteins known to interact with coated vesicles, were iodinated, placed in buffer, and incubated over the transfer for 1 h. After being rinsed extensively, the total amount of 125I associated with the filters was measured, and the filters were then processed for autoradiography. For alpha-actinin, the clathrin heavy chain and a series of lower molecular weight proteins were labeled. The binding of 125I alpha-actinin was inhibited with cold ligand and selectively released from the transfer with a buffer know to strip alpha-actinin from plasma membrane preparations. For 125I calmodulin the predominant binding site was also the clathrin heavy chain. Cold ligand inhibited binding and 60% of the detectable binding were calcium dependent. In addition, when these ligands were used in competition with each other, no significant inhibition was detected in the amount of binding associated with the clathrin heavy chain. These studies show that the clathrin heavy chain is a primary site of the clathrin cage receptive to intracellular interactions and furthermore suggest that the clathrin heavy chain consists of domains of biochemical specificity which may selectively affect the activities of coated vesicles.

Actinin↗
PubMed1999

Efficiency of four secondary enrichment protocols in differentiation and isolation of Listeria spp. and Listeria monocytogenes from smoked fish processing chains.

Four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h: Impedimetric method: Listeria electrical detection medium) were studied for their ability to isolate Listeria spp. and Listeria monocytogenes from fish and environmental samples collected along the processing chain of cold-smoked fish. From all methods, Listeria spp. and L. monocytogenes were respectively present in 56 and 34 of 315 samples analysed. Fraser broth incubated for 48 h gave the fewest false negative Listeria spp. results [4/56; (7.1%)], but concurrently only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes, Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp. positive samples. Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes positive samples correctly and gave fewer false positive results. The overall conclusion was that more than one isolation method is needed to accurately estimate L. monocytogenes contamination rates.

Animals↗
PubMed1991

Nucleotide sequence analysis of the V regions of two IgM cold agglutinins. Evidence that the VH4-21 gene segment is responsible for the major cross-reactive idiotype.

Cold agglutinins are human autoantibodies, usually of the IgM class, which agglutinate RBC at low temperature. The major subset recognizes the I/i carbohydrate Ag, and many of these antibodies bear cross-reacting idiotypic determinants. An anti-idiotypic mAb that is specific for one of the idiotopes largely confined to cold agglutinins has been used to identify and monitor tumor cells that secrete these molecules in two patients. The tumor cells were immortalized with EBV and the idiotope-positive lines used to investigate the utilization of the VH and VL genes by these antibodies. Nucleotide sequence analysis of the two cold agglutinins (FS-1 and FS-2) revealed the utilization of a single common gene segment, VH4-21. Serologic analysis documented that only human antibodies utilizing the VH4-21 gene segment were reactive in the idiotope assay, other VHIV antibodies as well as a panel of antibodies derived from other VH families being negative. The DH, JH, VK, and JK gene segments of FS-1 and FS-2 were structurally distinct. These data suggest that the structural basis for the cross-reactive idiotope as well as cold agglutinin activity is the VH4-21 gene segment. A nucleotide change in H chain CDR1 of both cold agglutinins results in the substitution of an aspartic acid residue for glycine at position 31, suggesting that this amino acid might be critical to recognition of the red cell Ag.

Aged↗
PubMed1992

VH restriction among human cold agglutinins. The VH4-21 gene segment is required to encode anti-I and anti-i specificities.

We previously reported that human autoantibodies with cold agglutinin activity contained a single human VH gene segment (VH4-21) which was also responsible for the cross-idiotypic specificity characteristic of the cold agglutinin response. To confirm and extend this observation we have analyzed at the nucleotide level the H and L chains of six new cold agglutinin molecules derived from different patients. We found that regardless of whether the antibody recognizes the i or the I red cell Ag, restriction at the VH gene segment level is absolute. We also found that even in the absence of somatic mutation the VH4-21 gene segment can encode both anti-i and anti-I specificities. Finally, although the VH4-21 gene segment is essential for cold agglutinin activity, the other genetic elements that contribute to the V region of the antibody molecules can be extremely diverse. The structural information provided in this report sharply restricts the requirement for encoding pathogenic cold agglutinin activity to one of the components of the H chain V region, specifically the VH gene segment. The implications of this apparently absolute requirement for a single VH gene segment, unprecedented in the human autoimmune response, are discussed.

Aged↗
PubMed2002

An Escherichia coli mutant lacking the cold shock-induced palmitoleoyltransferase of lipid A biosynthesis: absence of unsaturated acyl chains and antibiotic hypersensitivity at 12 degrees C.

An acyltransferase induced by cold shock in Escherichia coli, designated LpxP, incorporates a palmitoleoyl moiety into nascent lipid A in place of the secondary laurate chain normally added by LpxL(HtrB) (Carty, S. M., Sreekumar, K. R., and Raetz, C. R. H. (1999) J. Biol. Chem. 274, 9677-9685). To determine whether the palmitoleoyl residue alters the properties of the outer membrane and imparts physiological benefits at low growth temperatures, we constructed a chromosomal insertion mutation in lpxP, the structural gene for the transferase. Membranes from the lpxP mutant MKV11 grown at 12 degrees C lacked the cold-induced palmitoleoyltransferase present in membranes of cold-shocked wild type cells but retained normal levels of the constitutive lauroyltransferase encoded by lpxL. When examined by mass spectrometry, about two-thirds of the lipid A molecules isolated from wild type E. coli grown at 12 degrees C contained palmitoleate in place of laurate, whereas the lipid A of cold-adapted MKV11 contained only laurate in amounts comparable with those seen in wild type cells grown at 30 degrees C or above. To probe the integrity of the outer membrane, MKV11 and an isogenic wild type strain were grown at 30 or 12 degrees C and then tested for their susceptibility to antibiotics. MKV11 exhibited a 10-fold increase in sensitivity to rifampicin and vancomycin at 12 degrees C compared with wild type cells but showed identical resistance when grown at 30 degrees C. We suggest that the palmitoleoyltransferase may confer a selective advantage upon E. coli cells growing at lower temperatures by making the outer membrane a more effective barrier to harmful chemicals.

Acyltransferases↗
PubMed2001

Role of the chain termini for the folding transition state of the cold shock protein.

Residues Arg3 and Leu66 are crucially important for the enhanced stability of the cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus relative to its homologue Bs-CspB from the mesophile Bacillus subtilis. Arg3, which replaces Glu3 of Bs-CspB, accounts for two-thirds of the stability difference and for the entire difference in Coulombic interactions between the two proteins. Leu66, which replaces Glu66 of Bs-CspB, contributes additional hydrophobic interactions. To elucidate the role of these two residues near the chain termini for the rapid folding of the cold shock proteins, we performed an extensive mutational analysis of the folding kinetics to characterize interactions between residues 3, 46, and 66 in the transition state of folding. We employed a pressure-jump apparatus which allows folding to be followed over a broad range of temperatures and urea concentrations in the time range of microseconds to minutes. The N-terminal region folds early, and the interactions that originate from residue 3 are present to a large extent in the transition state already. They include a hydrophobic contribution, a general electrostatic stabilization by the positive charge of Arg3 in Bc-Csp, and a pairwise Coulombic repulsion with Glu46 in the Arg3Glu variant. The C-terminus appears to be largely unfolded in the transition state. The interactions of Leu66, including those with the already structured N-terminal region, are established only after passage through the transition state. The N- and C-termini of the cold shock proteins thus contribute differently to the folding kinetics, although they are very close in space in the folded protein.

Amino Acid Substitution↗
PubMed1988

Spectrotypic analysis of antibodies to insulin A and B chains.

Antibodies produced by immunization with native insulin were analyzed by isoelectric focusing for binding to isolated A and B chains. Antibodies to isolated A chain of beef insulin were found to have restricted spectrotypes and were seen after immunization with either beef or human insulin. Hyperimmunization with beef insulin, but not human, increased the heterogeneity of anti-A chain antibodies. Antibodies to isolated B chains were also electrophoretically restricted but showed less heterogeneity after hyperimmunization than anti-A chain responses. When binding to chains was carried out in the presence of excess cold insulin, anti-B chain spectrotypes were inhibited by the native molecule. In contrast, only a portion of anti-A chain spectrotypes were inhibited by native insulin, suggesting that these clonotypes are directed at epitopes not present on the surface of the molecule. These data indicate that the anti-insulin repertoire includes antibodies that can bind isolated chains as well as the native molecule. Some of the determinants on isolated A chain are not available on intact insulin and may arise from antigen catabolism.

Animals↗
PubMed1987

Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing.

Diluents containing sonicated liposomes of purified phosphatidylserine (PS), phosphatidylcholine (PC) with varying fatty acyl chain lengths and double bonds and cholesterol (CH) alone or in combination, or egg yolk lecithin were evaluated for protection of bull sperm during cold shock produced by rapid cooling from 25 to 0 degrees C and during freezing and thawing. Bull semen was washed twice and diluted to 50 X 10(6) sperm/ml in diluents containing no lipid, 0.5 or 5 mM sonicated lipid or 20% egg yolk and plunged into ice water to cold shock the sperm. Sperm so treated were frozen using conventional methods. The percentage of progressively motile sperm (MS) was estimated prior to cooling, after cold shock, and after freezing and thawing. Lipids with fatty acyl chains of less than 12 carbons were toxic to sperm cells. Phosphatidylserine alone or in combination with PC or CH, but not PC or CH alone, protected sperm from cold shock as well as did egg yolk lecithin liposomes or egg yolk. Liposomes of PS/PC or PS/CH were not better than PS in protecting sperm from cold shock. Lipid concentrations of 0.5 mM were more effective than liposomes at 5 mM in protecting sperm during freezing and thawing. During freezing, PS alone or in combination with PC partially protected sperm, but only PS/CH was as effective as egg yolk in protecting sperm from freeze-thaw damage. It is concluded that defined diluents, particularly those containing PS, may be useful in studies of cryobiology of spermatozoa.

Animals↗
PubMed2000

Application of the BAX for screening/genus Listeria polymerase chain reaction system for monitoring Listeria species in cold-smoked fish and in the smoked fish processing environment.

The cold-smoked fish industry was used as a model for the development of a system for monitoring Listeria spp. in foods and in the food processing environment. A total of 214 samples including raw fish, fish during the cold-smoking process, finished product, and environmental samples were collected from three processing facilities over two visits to each facility. Samples were screened for Listeria spp. using the BAX for Screening/genus Listeria polymerase chain reaction system (PCR) and by culture. Listeria spp., confirmed by the API Listeria test strip or by a PCR assay targeting the L. monocytogenes hlyA gene, were isolated from a total of 89 (41.6%) samples. Of these, 80 samples also tested positive for Listeria spp. using the BAX system. Specifically, 42 (55.3%) environmental samples (n = 76), 11 (25.6%) raw materials samples (n = 43), 20 (35.1%) samples from fish in various stages of processing (n = 57), and 7 (18.4%) finished product samples (n = 38) tested positive for Listeria spp. using the BAX system. Five (4.0%) of the 125 culture-negative samples yielded BAX system-positive results. Listeria isolates from each of nine culture-positive/BAX system-negative samples yielded a positive reaction when tested in pure culture by the BAX system, suggesting that our false-negative results were likely due to the presence of low Listeria numbers in the initial enrichment as opposed to nonreacting isolates. The employment of alternative enrichment protocols, such as the two-step enrichment recommended by the manufacturer, may increase the sensitivity of the assay.

Animals↗
PubMed1998

Polymerase chain reaction amplification of bacterial 16S rRNA genes from cold-cup biopsy forceps.

PURPOSE: In looking for a possible infectious cause for interstitial cystitis (IC), we previously determined that bladder tissue specimens from both IC patients and controls were uniformly positive by polymerase chain reaction assay (PCR) for bacterial 16S ribosomal RNA genes from various genera including Escherichia, Propionobacterium, Acinetobacter, and Salmonella. We therefore determined whether the biopsy forceps might be contaminated with bacterial DNA. MATERIALS AND METHODS: A total of 23 samples were obtained following disinfection of 6 cold-cup bladder biopsy forceps (2 to 5 specimens from each forceps over a period of 19 months). DNA was extracted from each sample, and PCR performed using nested primers from a highly conserved region of the bacterial 16S rRNA gene. Amplified DNA was purified and sequenced, and the sequences obtained were compared with bacterial rRNA gene sequences recorded in GenBank. RESULTS: Thirteen of 23 forceps specimens were positive by PCR for bacterial DNA, including at least one rinse from each of the 6 forceps. In comparison, none of 9 negative control specimens (sterile distilled water put into tubes and processed in the same manner as forceps rinses) had detectable bacterial DNA. Sequence data indicated the presence of a predominant organism in 12 of the 13 positive specimens, with >95% homology to DNA from several different genera of bacteria including Escherichia, Propionobacterium, Stenotrophomonas and Pseudomonas. CONCLUSIONS: These data indicate that reusable bladder biopsy forceps are frequently contaminated with bacterial DNA. Tissue specimens procured with such instruments therefore are inappropriate sources to look for the presence of bacterial pathogens by PCR.

Biopsy↗
PubMed1978

Identification of the sites in collagen alpha-chains that bind serum anti-gelatin factor (cold-insoluble globulin).

Anti-gelatin factor was prepared from guinea-pig and human serum by affinity chromatography on denatured type-I collagen. As shown previously, this component is related to cold-insoluble globulin. It reacted with 125I-labelled denatured collagen, and the reaction could be inhibited by preincubation with unlabelled collagenous components. In the inhibition assay comparable activities were observed for native and denatured type-I, -II, -III and -IV collagens. There was also no difference in reactivity between collagens of different species. The reactive sites in the collagen alpha-chains were located by inhibition assays on distinct CNBr- and collagenase-derived peptides. The results obtained with fragments from alpha1(I)-, alpha2- and alpha1(II)-chains indicate that the most active region is located between positions 643 and 819 of the alpha1-chain. Lower activities were found for other regions of collagen and may indicate that the factor has the potential to interact with several sites in the alpha-chains. The present data agree with observations by Kleinman, McGoodwin & Klebe [Biochem. Biophys. Res. Commun. (1976) 72, 426-432] on the specificity of a serum factor promoting the attachment of fibroblasts to collagen.

Animals↗
PubMed1994

Stage-specific requirement for myosin II during Dictyostelium development.

Dictyostelium cells that lack a functional myosin II heavy chain are motile and are capable of aggregation, but fail to undergo further multicellular development. We have used a Dictyostelium mutant expressing a cold-sensitive myosin heavy chain to examine the requirement for myosin throughout the course of development. The loss of myosin function upon cooling is rapid and reversible. Temperature-shift experiments reveal that myosin is essential during two different stages of development. During aggregation, myosin function appears to be necessary for cells to sort correctly in a way that allows further development to occur. During the final stage of development, it is required for the formation of a complete stalk and the raising of the spore head. Development between those stages, however, proceeds normally in the absence of myosin function. Aggregates at non-permissive temperature undergo an aberrant form of development resulting in a ball of cells. Calcofluor staining and reporter gene fusions reveal that these structures contain defective spores and a miniature stalk.

Animals↗
PubMed2000

Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes.

Listeria monocytogenes is a foodborne psychrotrophic pathogen that grows at refrigeration temperatures. Previous studies of fatty acid profiles of wild-type and cold-sensitive, branched-chain fatty acid deficient mutants of L. monocytogenes suggest that the fatty acid 12-methyltetradecanoic (anteiso-C(15:0)) plays a critical role in low-temperature growth of L. monocytogenes, presumably by maintaining membrane fluidity. The fluidity of isolated cytoplasmic membranes of wild-type (SLCC53 and 10403S), and a cold-sensitive mutant (cld-1) of L. monocytogenes, grown with and without the supplementation of 2-methylbutyric acid, has been studied using a panel of hydrocarbon-based nitroxides (2N10, 3N10, 4N10, and 5N10) and spectral deconvolution and simulation methods to obtain directly the Lorentzian line widths and hence rotational correlation times (tau(c)) and motional anisotropies of the nitroxides in the fast motional region. tau(c) values over the temperature range of -7 degrees C to 50 degrees C were similar for the membranes of strains SLCC53 and 10403S grown at 10 degrees C and 30 degrees C, and for strain cld-1 grown with 2-methylbutyric acid supplementation (which restores branched-chain fatty acids) at 30 degrees C. However, strain cld-1 exhibited a threefold higher tau(c) when grown without 2-methylbutyric acid supplementation (deficient in branched-chain fatty acids) compared to strains SLCC53, 10403S, and supplemented cld-1. No evidence was seen for a clear lipid phase transition in any sample. We conclude that the fatty acid anteiso-C(15:0) imparts an essential fluidity to the L. monocytogenes membrane that permits growth at refrigeration temperatures.

Anisotropy↗
PubMed1998

Risk factors for and prevention of sporadic infections with vero cytotoxin (shiga toxin) producing Escherichia coli O157.

BACKGROUND: Recent outbreaks of vero cytotoxin (shiga toxin) producing Escherichia coli O157 (VTEC O157) infection have stimulated debate on food safety. However, 90% of cases in England and Wales are sporadic. We report a case-control study of sporadic VTEC O157 infection. METHODS: We compared 85 sporadic cases of VTEC O157 infection, identified through population surveillance, with 142 controls, randomly selected from general practitioners' lists. We matched cases and controls for age, sex, and family doctor's practice. Exposures to foods, water, animals, farms, and environmental factors were recorded. We visited the premises concerned when cases had eaten beefburgers or cooked sliced meats from caterers or had had contact with a farm. FINDINGS: Consumption of a beefburger from a catering premises other than from a fast-food chain A (a national chain) and consumption of cold cooked sliced meat (eg, in a salad or sandwich) from caterers, but not butchers, was associated with VTEC O157 infection (odds ratios 4.63 [95% CI 1.33-30.14] and 3.36 [1.04-12.74], respectively). Policies for ensuring thorough cooking of burgers by one national fast-food chain differed from the other catering premises we visited. There was evidence of person-to-person spread and transmission of VTEC O157 infection from animals. INTERPRETATION: Local inspection of catering establishments that serve cooked meats together with public education to prevent spread on farms and in houses would reduce the burden of VTEC O157 infection by about 10% for each risk factor.

Agriculture↗
PubMed1976

Some correlations between specificity and sequence of the first complementarity-determining segments of human kappa light chains.

Examination of the sequences of the first complementarity-determining segments of the light chains of two IgM cold agglutinins agains blood group I, four monoclonal IgM antibodies against IgG proteins, and of three Bence Jones proteins provides clues for predicting which residues contribute to antibody specificity and indicates that these predictions may be tested by evaluating recovery of antibody activity and specificity when various light chains are recombined with homologous and heterologous heavy chains.

Antibodies, Anti-Idiotypic↗
PubMed1994

Metabolism of the Raffinose Family Oligosaccharides in Leaves of Ajuga reptans L. (Cold Acclimation, Translocation, and Sink to Source Transition: Discovery of Chain Elongation Enzyme).

Ajuga reptans is a frost-hardy, perennial labiate that is known for its high content of raffinose family oligosaccharide(s) (RFO). Seasonal variations in soluble nonstructural carbohydrate levels in above-ground parts of Ajuga showed that the RFO were by far the most predominant components throughout the whole year. RFO were lowest in summer (75 mg/g fresh weight) and highest in fall/winter (200 mg/g fresh weight), whereas sucrose and starch were only minor components. Cold treatment (14 d at 10/3[deg]C, day/night) of plants that were precultivated under warm conditions (25[deg]C) lowered the temperature optimum of net photosynthesis from 16[deg] to 8[deg]C, decreased the maximum rate, and increased the total nonstructural carbohydrate content of leaves by a factor of about 10, mainly because of an increase of RFO. The degree of polymerization of the RFO increased sequentially up to at least 15. A novel, galactinol-independent galactosyltransferase enzyme was found, forming from two molecules of RFO, the next higher and lower degree of polymerization of RFO. The enzyme had a pH optimum of 4.5 to 5.0 and may be responsible for RFO chain elongation. RFO were the main carbohydrates translocated in the phloem, with stachyose being by far the most dominant form. Studies of carbon balance during leaf development revealed a transition point between import and export at approximately 25% maximal leaf area. RFO synthesis could be detected even before the commencement of export, suggesting the existence of a nonphloem-linked RFO pool even in very young leaves. Taken together, it seems that Ajuga leaves contain two pools of RFO metabolism, a pronounced long-term storage pool in the mesophyll, possibly also involved in frost resistance, and a transport pool in the phloem.

Journal Article↗