Comparative assessment of inorganic pyrophosphate and pyrophosphatase levels of Escherichia coli, Clostridium pasteurianum, and Clostridium thermoaceticum

Abstract Pyrophosphatase (PP_iase) specific activities were much higher in anaerobic cultures of Escherichia coli (0.54 units) than in Clostridium pasteurianum (0.067 units) and Clostridium thermoaceticum (0.017 units) (1 unit = 1 μ mole PP_i hydrolyzed/min per mg cell dry wt.), and were fairly constant throughout the growth of all three organisms. Conversely, intracellular levels of pyrophosphate (PP_i) were very low and constant in E. coli throughout growth (0.3 mM), while those of C. pasteurianum and C. thermoaceticum were higher (1.44 and 0.8 mM, respectively) and peaked sharply during mid log-phase of growth. PP_iase and intracellular PP_i remained relatively constant in E. coli when grown aerobically or anaerobically, and when growth was in medium containing PP_i as the sole source of supplemental phosphorus.     

Congenital heart disease in the offspring and maternal habits and home exposures during pregnancy.

To test the effect of maternal habits and home exposures during early pregnancy on the occurrence of congenital heart disease in the offspring, 406 cases and 756 controls were studied. The cases included all cardiovascular malformations detected in Finland during 1982-1983, while the healthy controls were randomly selected from all babies born during the same period. Case and control mothers were interviewed after delivery using a structured and pre-tested questionnaire. Maternal overall drug consumption during the first trimester was as prevalent among case mothers (13.3%) as controls (14.6%). Neither was the risk of congenital heart disease associated with maternal use of contraceptive pills, salicylates, diazepam, or sweetening agents separately. Maternal exposures to disinfectants, dyes, lacquers, paints, pesticides, or glues at home were equally prevalent in case and control groups. Several earlier miscarriages was a predictor of an infant born with congenital heart disease (OR = 2.7, CI95 = 1.4-5.3). Maternal ultrasound examination was performed during the first 16 weeks of pregnancy more often among the case group (28.3%) than among the control group (22.0%). However, the association between ultrasound examination and the risk of congenital heart disease in the offspring was not statistically significant (OR = 1.2, 95% confidence interval 0.9-1.7) when adjusted for confounding factors such as the threat of miscarriage in logistic regression analysis. It is concluded that maternal ultrasound examination, intake of some common drugs, and exposure to a number of environmental factors at home during early pregnancy are probably not harmful for the developing fetal heart.     

Sequence heterogeneity in the duplicate large subunit ribosomal RNA genes of Tetrahymena pyriformis mitochondrial DNA

In the ciliated protozoan, Tetrahymena pyriformis, the mitochondrial large subunit ribosomal RNA (LSU rRNA) is discontinuous, consisting of two discrete RNA species: a 280-nucleotide LSU alpha (constituting the 5'-portion) and a 2315-nucleotide LSU beta (corresponding to the remaining 3'-portion of this rRNA). The T. pyriformis mitochondrial genome contains two copies of the LSU alpha.beta gene complex, and we have previously provided evidence that both copies are transcribed (Heinonen, T. Y. K., Schnare, M. N., Young, P. G., and Gray, M. W. (1987) J. Biol. Chem. 262, 2879-2887). We now report the complete sequences of the two copies of the LSU alpha.beta gene complex. These are not identical, but differ at 5 out of the 2595 positions by single nucleotide substitutions in one sequence relative to the other. In the secondary structure model we propose here, two of these differences are located in base-paired regions of the LSU rRNA; however, they do not interrupt the complementary interactions in these helices. The other three differences occur in single-stranded regions of the secondary structure. The base substitutions documented here are not localized to those regions of LSU rRNA that are the most highly conserved in global phylogenetic comparisons, and therefore it seems unlikely that they are of fundamental functional significance. Whether they might exert more subtle effects on ribosome function remains to be determined.     

Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12.

Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced. The 5' end of the ppa mRNA was identified by primer extension mapping. A typical E. coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end. The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced. The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P. Burton, D. C. Hall, and J. Josse, J. Biol. Chem. 245:4346-4351, 1970). Furthermore, the partial amino acid sequence (residues 1 to 108) of E. coli PPase determined by S. A. Cohen (Ph.D. thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence. This is the first report of the cloning of a PPase gene.     

Variation in birch bark secondary chemistry between and within clones: implications for herbivory by hares

We studied the variation in bark chemistry between and within 19 European white birch ( Betula pendula ) clones and its implications to resistance to the mountain hare ( Lepus timidus ). We used one-year-old clonal plantlets originating from randomly selected naturally regenerated parental trees. The same clones were used in both chemical analyses and in feeding experiments. The condensed tannins were analysed by an acid butanol assay, other phenolic components by HPLC-DAD, and triterpenoid components by HPLC-MS. The resistance to hare was tested in open-field feeding experiment. The main phenolic compounds in birch bark were catechin derivatives, rhododendrin, platyphylloside, and condensed tannins, and the main triterpenoids were papyriferic acid and pendulic acid. Most of the variation in the concentrations of the studied compounds was found between clones for the studied phenolics and large variation for triterpenoid components were found both between clones and among plantlets within the same clone. Hares clearly selected among the studied clones. Our results suggest that birch bark chemistry play an important role in resistance to herbivory by hare. The total triterpenoids and total flavonoid-aglycones showed significant negative correlation with hare feeding. It seems that a genetic basis for bark chemistry and birch resistance is strong. Such a high variation in secondary chemistry both between clones and within individual clones indicates that European white birch populations have a good resistance towards variable environmental conditions and varying pressures from herbivory.     

Characterization of the 5' flanking region of the Escherichia coli ppa gene encoding inorganic pyrophosphatase: mutations in the ribosome-binding site decrease the level of ppa mRNA.

We have previously cloned and sequenced the ppa gene, encoding inorganic pyrophosphatase (PPase), of Escherichia coli K12 [Lahti, R., Pitk?ranta, T., Valve, E., Ilta, I., Kukko-Kalske, E. & Heinonen, J. (1988) Journal of Bacteriology 170, 5901-5907]. In this work mutations were constructed in the 5' flanking region of E. coli ppa and the effect on expression was determined. The minimum length of the fully active ppa5' flanking region was shown to be 117 bp. Further deletion decreased the activity, and upon deletion to nucleotide -37 the promoter activity was totally lost. A clear point of inflection was observed in the inactivation upon deletion over the nucleotide -50. This is consistent with the fact that by binding to promoters RNA polymerase holoenzyme generally covers the -50 to +20 region in E. coli genes. When the -35 sequence of ppa, AAGACA, was mutated to AAAACA, ppa expression, as measured by PPase production, decreased to 20% of the wild-type, whereas by the change of the -10 sequence, TATAAT, to TTTAAT or TATAAA, the ppa gene was totally inactivated. Furthermore, when the ribosome-binding site (RBS) sequence, AGGAAA, was altered to AAGAAA, PPase production decreased to 19% of the wild-type. Surprisingly, when the RBS sequence was mutated to the consensus RBS sequence, AGGAGG, the intracellular levels of both ppa mRNA and PPase decreased drastically. The implications of these results are discussed.     

Food-habitat relationship of harbour seals and black cormorants in Skagerrak and Kattegat

Comparative studies on the food of the black cormorant Phalacrocorax carbo and the harbour seal Phoca vitulina were carried out in two contrasting habitat types. Both species feed predominantly on bottom-dwelling fish. Black cormorants are able to utilize fish found both on vegetation-covered and naked sea beds at a water depth of less than 10 m, while harbour seals feed mainly on soft sea bottoms above 30 m where vegetation is scarce or lacking. The food overlap of the two piscivores is large in environments where large shallow sea bottoms are available, and small in areas where sea bottoms of soft material is found at greater depth. In areas where the shallow bottoms are covered by vegetation only, cormorants are able to feed on the fish species inhabiting this kind of environment. There is no evidence that these predators make a choice regarding prey species.     

N-glycomic Profiling as a Tool to Separate Rectal Adenomas from Carcinomas

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world''s three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I–II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.Glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans, that cover all human cells. Around 1% of the human genome participates in the biosynthesis of glycans(1). This biosynthesis is the most complex post-translational modification of proteins, and the great variability in glycan structures contains a tremendous ability to fine-tune the chemical and biological properties of glycoproteins. The glycosylation process occurs most abundantly in the Golgi apparatus and the endoplasmic reticulum, but also occurs in the cytoplasm and nucleus (2). Most glycoconjugates are localized to cell surfaces, where glycans participate in events essential for cell viability and function, such as cell adhesion, motility, and intracellular signaling (2). Changes in these functions are key steps seen when normal cells transform to malignant ones, and these are also reflected in changes of a cell''s glycan profile, observed in many cancers (3, 4). Specific structural changes in glycans may serve as cancer biomarkers (5, 6), and changes in glycosylation profiles are related to aggressive behavior in tumor cells (7–9).Cancer-associated asparagine-linked glycan (N-glycan) structures may play specific roles in supporting tumor progression; growth (10, 11), invasion (12, 13), and angiogenesis (14). Changes in the N-glycan profile emerge in numerous cancers, including lung (15, 16), breast (17), and colorectal cancer (CRC)¹ (16, 18). Balog et al. (18) comparing the N-glycomic profile of CRC tissue to adjacent normal mucosa, reported differences in specific glycan structures. Moreover, serum N-glycosylation profile from patients with CRC differ from those of healthy controls (19).Colorectal cancer is the third most common cause of cancer-related death worldwide and its incidence is rising; 40% of CRCs are of rectal origin. Roughly 40% of patients have localized disease (stage I–II; Dukes A–B), another 40% loco regional disease (stage III; Dukes C), and 20% metastasized disease (stage IV; Dukes D) (20). Although stage at diagnosis is the most important factor determining prognosis, clinical outcome, and response to adjuvant treatment can markedly vary within each stage. Adjuvant therapy routinely goes to stage III patients, but the benefit of adjuvant treatment for stage II patients is unclear. Of stage II patients, 80% are cured by radical surgery alone. To identify patients who will benefit from postoperative treatment, we need novel biomarkers. The glycan profile of the tumor tissue could provide new biomarkers for diagnosis and prognosis of cancer.In this study, we characterized the N-glycomic profiles of rectal adenomas and carcinomas by MALDI-TOF mass spectrometric (MS) profiling of asparagine-linked glycans. Our aim was to identify differences between adenomas and carcinomas, and also between cancers of different stages. Based on glycan profiling, we also chose, for immunohistochemical expression studies of a series of 220 CRC patients, two glycan markers: sialyl Lewis a and pauci-mannose.