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PubMed1998

A novel plasmid shuttle vector for the detection and analysis of microsatellite instability in cell lines.

Microsatellite instability is an important feature of tumors from hereditary nonpolyposis colorectal carcinoma (HNPCC) patients as well as a variety of sporadic tumors. Here, we present a novel plasmid shuttle vector for the detection of this replication error (RER+) phenotype in human cell lines. The episomely replicated plasmid pZCA29 harbours the bacterial beta-galactosidase gene interrupted by two palindromically arranged poly-(CA)-repeat tracts. The resulting + 1-frameshift leads to white colonies of Escherichia coli DH10B on X-Gal/IPTG1 agar plates. Mutations in the repeats characteristic of the RER+-phenotype may result in the loss or gain of CA-repeats leading to blue bacterial colonies. We transiently transfected the colorectal cancer cell lines SW480 and HCT116 with the plasmid pZCA29, isolated replicated plasmid DNA after several days and used it to transform E. coli DH10B. We found 1.0 to 1.7% blue colonies after passage of the plasmid through the RER+-cell line SW480 in contrast to 3.5 to 8.1% blue colonies after transfection of the RER+-cell line HCT116, the mutation frequencies increasing with incubation time. Sequence analysis of mutated plasmids revealed mostly 2-bp deletions which occurred especially in one of the repeat tracts. We conclude that pZCA29 appears to be a suitable shuttle vector for the detection and analysis of a RER+-phenotype in cell lines.

Colony Count, Microbial↗
PubMedDate not supplied

Characterization of 64-, 123- and 182-base-pair exons in the mouse alpha 2(IV) collagen gene.

Genes encoding types I, II and III collagens (fibrillar collagens) contain many discrete-size exons, most of them 54 base pairs (bp) long, in addition to the 45-, 99-, 108- and 162-bp exons. It has been suggested that these collagen genes evolved from an ancestral coding unit of 54 bp. Type IV collagen is a specific component of basement membranes and contains two genetically distinct polypeptides, the alpha 1(IV) and alpha 2(IV) chains. It differs from the types I-III collagens in that it contains interruptions in the Gly-X-Y repeat sequence and does not form ordered fibrillar structures. We have isolated complementary DNA and genomic clones for the mouse alpha 2(IV) collagen chain and here characterize 64-, 123- and 182-bp exons in the Gly-X-Y coding domain of the gene. The data suggest that the alpha 2(IV) collagen gene may have evolved differently from those encoding the fibrillar collagens.

Animals↗
PubMed1985

Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system.

Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A- and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide.

Base Sequence↗
PubMed1994

Effects of acute exercise on lymphocyte subsets and metabolic activity.

Lymphocyte subsets, their responsiveness to mitogen and their capacity for glutamine oxidation and glycolysis were assessed in seven subjects before and after an acute bout of interval exercise, the purpose being to establish whether exercise is associated with alterations in lymphocyte metabolic capacities. The subjects exercised at 112% of their maximal work capacity (as determined by pre-test) on a treadmill and performed 25 repeat tests, each of 1 min duration interrupted by 2 min rest periods. Venous blood samples were taken at rest and 3 min following completion of exercise. Acute exercise was associated with significant decreases in the percentage of T- (p < 0.01) and B-cells (p < 0.01) and an increase in the percentage of NK-cells (p < 0.05). These changes were accompanied by a significant decrease in the responsiveness of peripheral blood lymphocytes to the mitogen concanavalin A (p < 0.05). Acute exercise was also associated with profound changes in the metabolic capacities of peripheral blood lymphocytes: rates of 14CO2 production from [U-14C]glutamine (19%: p < 0.05) and lactate (27%: p < 0.05) production were increased significantly in response to interval exercise. Linear regression analysis revealed significant correlation between the exercise-mediated changes (%) in T- and NK-cells and changes (%) in both lymphocyte responsiveness to concanavalin A and metabolic capacity, particularly glutamine oxidation to CO2. One interpretation of these data is that acute exercise promotes a redistribution in lymphocyte subsets, and that it is this redistribution that is the basis of both the impairment in lymphocyte responsiveness to mitogens and the increase in lymphocyte metabolic capacity, especially glutamine oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2001

Modulation of myosin function by isoform-specific properties of Saccharomyces cerevisiae and muscle tropomyosins.

Tropomyosin is an extended coiled-coil protein that influences actin function by binding longitudinally along thin filaments. The present work compares cardiac tropomyosin and the two tropomyosins from Saccharomyces cerevisiae, TPM1 and TPM2, that are much shorter than vertebrate tropomyosins. Unlike cardiac tropomyosin, the phase of the coiled-coil-forming heptad repeat of TPM2 is discontinuous; it is interrupted by a 4-residue deletion. TPM1 has two such deletions, which flank the 38-residue partial gene duplication that causes TPM1 to span five actins instead of the four of TPM2. Each of the three tropomyosin isoforms modulates actin-myosin interactions, with isoform-specific effects on cooperativity and strength of myosin binding. These different properties can be explained by a model that combines opposite effects, steric hindrance between myosin and tropomyosin when the latter is bound to a subset of its sites on actin, and also indirect, favorable interactions between tropomyosin and myosin, mediated by mutually promoted changes in actin. Both of these effects are influenced by which tropomyosin isoform is present. Finally, the tropomyosins have isoform-specific effects on in vitro sliding speed and on the myosin concentration dependence of this movement, suggesting that non-muscle tropomyosin isoforms exist, at least in part, to modulate myosin function.

Actins↗
PubMed2004

Localization of the serine protease-binding sites in the collagen-like domain of mannose-binding protein: indirect effects of naturally occurring mutations on protease binding and activation.

Mutations in the collagen-like domain of serum mannose-binding protein (MBP) interfere with the ability of the protein to initiate complement fixation through the MBP-associated serine proteases (MASPs). The resulting deficiency in the innate immune response leads to susceptibility to infections. Studies have been undertaken to define the region of MBP that interacts with MASPs and to determine how the naturally occurring mutations affect this interaction. Truncated and modified MBPs and synthetic peptides that represent segments of the collagen-like domain of MBP have been used to demonstrate that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. The binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical. The two most common naturally occurring mutations in MBP result in substitution of acidic amino acids for glycine residues in Gly-X-Y triplets on the N-terminal side of the hinge. Circular dichroism analysis and differential scanning calorimetry demonstrate that the triple helical structure of the collagen-like domain is largely intact in the mutant proteins, but it is more easily unfolded than in wild-type MBP. Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs. In addition, at least one of the mutations has a further effect on the ability of MBP to activate MASPs.

Amino Acid Sequence↗
PubMed1976

Parasympathetic innervation and pancreatic secretion: the role of the gastric antrum.

The role of the antrum on vagally mediated pancreatic secretion was studied in 8 conscious dogs prepared with chronic pancreatic and gastric fistulae. After completion of control studies 6 were subjected to antrectomy and 2 to antroneurolysis (to interrupt submucosal nerve connections); secretory studies were repeated. With the animals secreting in response to secretin(0.03 u per kg-min) or secretin with cholecystokinin (0.05 u per kg-min), the following were administered: 1) insulin 0.2 u/kg; 2) atropine 0.2 and 0.4 mg/kg; 3) insulin after atropine. Insulin hypoglycemia elicited a marked enzyme response. Both antrectomy and antroneurolysis markedly reduced (80%) the enzyme response to insulin hypoglycemia. Atropine 0.2 mg/kg abolished the insulin response and at 0.4 mg/kg inhibited (50%) the enzyme response to cholecystokinin; these effects were unaltered by antrectomy or antroneurolysis. These experiments suggest that the pancreatic enzyme response to insulin hypoglycemia is predominantly mediated through the vagal release of antral gastrin. Furthermore, antrectomy and antroneurolysis do not affect the enzyme response to cholecystokinin nor do they alter the inhibitory effects of atropine. The inhibitio- by atropine suggests that a cholinergic background exerts a permissive effect on CCK-mediated enzyme secretion.

Animals↗
PubMed2000

A comparison of lactate concentration in plasma collected from the toe, ear, and fingertip after a simulated rowing exercise.

OBJECTIVE: To examine the validity of using blood taken from the toe for the assessment of plasma lactate concentration in rowers. To achieve this, values were compared with those taken from the fingertip and earlobe. METHODS: Nine subjects exercised at two separate submaximum workloads on the Concept II rowing ergometer. The loads, each lasting four minutes, elicited mean (SD) heart rate responses of 160.1 (8.5) and 180.1 (5.7) beats/min, which corresponded to 76.4 (6.1)% and 91.9 (4.7)% of the estimated heart rate maximum of the subjects. Blood was simultaneously removed after the cessation of exercise by three experimenters and was analysed for plasma lactate concentration. RESULTS: At 76.4% of estimated heart rate maximum, the mean (SD) plasma lactate concentrations sampled from the fingertip, toe, and earlobe were 6.36 (1.58), 5.81 (1.11), and 5.29 (1.24) mmol/l respectively. At 91.9% of estimated heart rate maximum, respective values were 8.81 (2.30), 8.53 (1.37), and 8.41 (2.35) mmol/l. No significant differences (p > 0.05) were found between any of the sites at either work intensity. CONCLUSIONS: The toe may offer a practical alternative for assessing the concentration of lactate during rowing, having the advantage that repeated blood samples can be removed without interruption of the rowing action.

Adult↗
PubMed1980

Regular and irregular cycles of interdigestive contractions in the stomach.

We have previously reported that during the interdigestive state repeated episodes of high-amplitude contractions interrupted by long-lasting motor quiescence were seen to occur regularly until the next meal in the dog. However, none of the dogs used in our laboratory showed such regular changes in gastric motor pattern all the time during the interdigestive state. In the present study, we measured plasma motilin concentrations and intraduodenal pH together with gastric motor activity in conscious dogs. It was found that as long as intraduodenal pH remained between 7.0 and 8.5 the regular occurrence of the interdigestive contractions was closely associated with increases in the plasma immunoreactive motilin (IRM) concentration. However, when intraduodenal pH became acid, there were no typical interdigestive contractions even though plasma IRM concentration was elevated. We consider that high-amplitude contractions like the interdigestive contractions could not be evoked in the acid-secreting stomach even if the plasma IRM concentration was elevated during the interdigestive state. Acid secretion during the fasted state, the cause of which is not known, seems to be one of the main factors in disturbing the regularity of the interdigestive motor pattern.

Animals↗
PubMed1990

A case of pseudo-Bartter's syndrome associated with hypergastrinemia, thrombocytosis and increased serum thyroxine-binding globulin.

A housewife, 40 years of age, was admitted with dysesthesia of the extremities, muscle weakness, and attacks of adynamia and thirst. She had been taking a laxative for more than 20 years. On physical examination, blood pressure was 94/56 mmHg. Laboratory tests revealed thrombocytosis, low serum K and marked increases in both plasma renin activity and serum aldosterone. Serum TBG was increased. Serum gastrin was also markedly increased and could not be enhanced by exogenous secretin. Both angiotensin 11 loading test and noradrenalin loading test failed to increase blood pressure. Ammonium chloride loading to examine the disturbance of urinary acidification was abnormal in the short term test and borderline in the long term test. Following a diagnosis of pseudo-Bartter's syndrome induced by long term intake of laxative and repeated diarrhea, the administration of laxative was interrupted and potassium, indomethacin and spironolactone were administered. However, serum K remained low. Hypergastrinemia, thrombocytosis and a high serum TBG level also persisted, the causes of which remain unknown. This is the first reported case of pseudo-Bartter's syndrome associated with hypergastrinemia, thrombocytosis and increased serum TBG.

Adult↗
PubMed1992

Post-cholecystectomy diarrhea: evidence of bile acid malabsorption assessed by SeHCAT test.

Although bile acid malabsorption (BAM) in post-cholecystectomy diarrhea (PCD) is a well-known clinical condition, its true etiopathogenetic role is not entirely clear. The SeHCAT (23-selena-25-homotaurocholic acid) test, a simple and reliable BAM test, was performed in 33 cholecystectomized patients, 26 with chronic diarrhea. The test revealed a marked degree of BAM in 25/26 cases. Cholestyramine in doses of 2-12 g/day was effective in 23/25, ineffective in two, and was not tolerated in one patient. When treatment was suspended, diarrhea recurred in nine, whereas bowel habit remained regular in 60%, with brief sporadic episodes of diarrhea in the other cases. The SeHCAT test was repeated in 11 cases after cholestyramine treatment interruption, and revealed the normalization of parameters in two patients and an improvement in three. We conclude that BAM is an important etiopathogenetic factor in PCD that responds favorably to cholestyramine. In 60% of the cases, it resolved diarrhea definitively, although without eliminating BAM in all cases: this suggests that existence of other factors associated with BAM. The SeHCAT test is essential for a differential diagnosis between PCD and the irritable bowel syndrome.

Adult↗
PubMed1989

The complete primary structure for the alpha 1-chain of mouse collagen IV. Differential evolution of collagen IV domains.

We report here the complete nucleotide and amino acid sequences for the alpha 1-chain of mouse collagen IV which is 1669 amino acids in length, including a putative 27-residue signal peptide. In comparison with the amino acid sequence for the alpha 2-chain (Saus, J., Quinones, S., MacKrell, A. J., Blumberg, B., Muthkumaran, G., Pihlajaniemi, J., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6318-6324), the two chains of collagen IV are 43% identical. Most of the interruptions of the Gly-X-Y repeat are homologously placed but strikingly show no sequence similarity between the two chains. Availability of the amino acid sequences for human collagen IV allows a detailed comparison of the primary structure of collagen IV and reveals evolutionarily conserved domains of the protein. Between the two species, the alpha 1 (IV) chains are 90.6% and the alpha 2 (IV) chains are 83.5% identical in sequence. We discuss these data with respect to differential evolution between and within the collagen IV chain types.

Amino Acid Sequence↗
PubMed1989

The complete primary structure of mouse alpha 2(IV) collagen. Alignment with mouse alpha 1(IV) collagen.

We have determined the nucleotide and amino acid sequences of mouse alpha 2(IV) collagen which is 1707 amino acids long. The primary structure includes a putative 28-residue signal peptide and contains three distinct domains: 1) the 7 S domain (residues 29-171), which contains 5 cysteine and 8 lysine residues, is involved in the cross-linking and assembly of four collagen IV molecules; 2) the triple-helical domain (residues 172-1480), which has 24 sequence interruptions in the Gly-X-Y repeat up to 24 residues in length; and 3) the NC1 domain (residues 1481-1707), which is involved in the end-to-end assembly of collagen IV and is the most highly conserved domain of the protein. Alignment of the primary structure of the alpha 2(IV) chain with that of the alpha 1(IV) chain reported in the accompanying paper (Muthukumaran, G., Blumberg, B., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6310-6317) suggests that a heterotrimeric collagen IV molecule contains 26 imperfections in the triple-helical domain. The proposed alignment is consistent with the physical data on the length and flexibility of collagen IV.

Amino Acid Sequence↗
PubMed1983

[Distribution of salicylic acid and hydrolysis of acetylsalicylic acid in the human fetus in early pregnancy].

The materno-fetal transfer of salicylic acid and its distribution in the fetal organism was investigated in women of early pregnancy. Acetylsalicylic acid (Acesal) was administered orally in a single dose or in repeated doses at different times before legal interruption. The mean passage rates were about 6-15%. They were independent of the maternal serum concentrations of salicylic acid. The distribution of salicylic acid on the fetal liver, intestine, kidneys, lungs and brain was different. All fetal organs (9th to 15th week of gestation) studied exhibit an acetylsalicylic acid-splitting esterase activity. The esterase activity of the fetal liver was about 30% of the hydrolytic activity of the adult liver. The esterase activity was mainly located in the 105 000 X g-supernatant of cell homogenates.

Aspirin↗
PubMed1978

Temperature cycling preserves platelet shape and enhances in vitro test scores during storage at 4 degrees.

Platelets stored at 4 degrees develop morphological changes which precede decreases in in vitro aggregation and recovery from hypotonic shock. Platelets maintained at 4 degrees for 60 min lack microtubules and lose their discoid shape as a result of the formation of pseudopodia. Exposure to continuous cold for 24 hr causes irreversible platelet shape changes and induces spontaneous aggregation. Quantitative electron microscopic analysis of platelet microtubules showed that platelets lost their ability to reassemble microtubules after 24 hr of continuous storage at 4 degrees. However, interruption of continuous cold storage with short, repeated pulses of 37 degrees (temperature cycling) extended microtubule reassembly capabilities for 72 hr and maintained platelet shape change capabilities. A comparison of platelets stored continuously at 4 degrees with those stored by temperature cycling showed that temperature-cycled platelets possessed improved aggregation responses and significantly improved recoveries from hypotonic shock. These data indicate that 37 degrees reactivation pulses during cold storage provide platelets with markedly improved morphology and in vitro test scores and suggest that temperature cycling may be used to extend blood bank storage of platelets.

Blood Platelets↗
PubMed1994

Complete primary structure of the human type IV collagen alpha 4(IV) chain. Comparison with structure and expression of the other alpha (IV) chains.

The entire sequence of the human alpha 4(IV) collagen chain was determined from cDNA clones and polymerase chain reaction-amplified DNAs. The complete translation product has 1,690 amino acid residues and the processed alpha 4(IV) chain proper 1,652 residues. There is a 38-residue putative signal peptide, a 1,421-residue collagenous domain starting with a 23-residue noncollagenous sequence, and a 231-residue NC1 domain. The Gly-Xaa-Yaa-repeat sequence of the collagenous domain is interrupted at 26 locations by noncollagenous sequences of 1-12 residues in length. The alpha 4(IV) chain contains 31 cysteine residues of which 18 are conserved in the other type IV collagen alpha chains. The calculated molecular weight of the mature alpha 4(IV) chain is 164,123. Analysis of the primary structure showed that the alpha 4(IV) chain belongs to the alpha 2-like type IV collagen chains together with alpha 2(IV) and alpha 6(IV). Northern analyses with RNA from several human fetal tissues revealed quite similar expression patterns for the alpha 4(IV) and alpha 3(IV) chains, but there were also distinct differences in some tissues. The expression patterns of alpha 5(IV) and alpha 6(IV) differed extensively between each other and they also differed from those of alpha 3(IV) and alpha 4(IV).

Amino Acid Sequence↗
PubMed1994

Complete primary structure of the human alpha 3(IV) collagen chain. Coexpression of the alpha 3(IV) and alpha 4(IV) collagen chains in human tissues.

We report the entire primary structure of the human alpha 3(IV) collagen chain determined from cDNA clones and polymerase chain reaction-amplified DNAs. The deduced amino acid sequence demonstrates that the complete translation product consists of 1670 amino acid residues and the mature alpha 3(IV) chain contains 1642 residues with a corresponding calculated molecular mass of 161,753. The full-length translated polypeptide has a signal peptide of 28 amino acids, a 1410-residue collagenous domain starting with a 14-residue noncollagenous sequence, and a 232-residue NC1 domain. There are 23 noncollagenous interruptions in the Gly-X-Y repeat sequence of the collagenous domain. The major transcription start site of the alpha 3(IV) chain gene was also determined from genomic DNA by primer extension and S1 nuclease protection assays. Northern analysis revealed coexpression of the alpha 3(IV) and alpha 4(IV) chains in tissues where expression was observed such as in kidney, muscle, and lung.

Amino Acid Sequence↗
PubMed1996

Novel germline p16 mutation in familial malignant melanoma in southern Sweden.

The p16 (CDKN2/MTS1/INK4a) malignant melanoma susceptibility gene was analyzed in 10 melanoma kindreds from southern Sweden using single-stranded conformation polymorphism analysis of all three exons and flanking intron regions followed by sequence analysis. A novel germline mutation, constituting an in-frame 3-bp duplication at nucleotide 332 in exon 2, was identified in two families (Lund M2 and M9). The mutation results in an insertion of Arg at codon 105, which interrupts the last of the four ankyrin repeats of the p16 protein, motifs which have been demonstrated as important in binding and inhibiting the activity of cyclin D-dependent kinases 4 and 6 in cell cycle G1 phase regulation. All five tested individuals of Lund M2 and M9 affected by melanoma were mutation carriers, as were five melanoma-free individuals. Other malignancies observed in gene carriers or obligate carriers included cervical, breast, and pancreatic carcinomas and a non-Hodgkin's lymphoma. Analysis of microsatellite markers adjacent to the p16 gene at chromosomal region 9p21 revealed that both families share a common haplotype, in keeping with a common ancestor.

Carrier Proteins↗