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Timo J Nevalainen

Publications and source records attributed to Timo J Nevalainen.

17 recordsLinked to original sources
PubMed2006

Analysis of expression of secreted phospholipases A2 in mouse tissues at protein and mRNA levels.

Secreted phospholipases A(2) (sPLA(2)) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA(2)s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA(2)s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA(2)s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA(2) proteins corresponded to the expression of sPLA(2)s at the mRNA level. With a few exceptions, the sPLA(2) proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA(2) is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA(2) in the intestine of GIIA sPLA(2)-deficient C57BL/6J mice is not compensated by other sPLA(2)s under normal conditions. Outside the gastrointestinal tract, sPLA(2)s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA(2) proteins in mouse serum and tissues in various experimental settings.

Animals↗
PubMed2006

Antibacterial effects of human group IIA and group XIIA phospholipase A2 against Helicobacter pylori in vitro.

Group IIA phospholipase A2 (PLA2-IIA) is an enzyme which has important roles in inflammation and infection. Recently, a novel human secretory PLA2 called group XIIA PLA2 (PLA2-XIIA) has been identified. Both PLA2-IIA and PLA2-XIIA are bactericidal against Gram-positive bacteria like many other secretory PLA2s. However, PLA2-XIIA is the only known PLA2 displaying significant bactericidal activity against the Gram-negative bacterium Escherichia coli. We examined the antibacterial properties of recombinant human PLA2-IIA and PLA2-XIIA against Helicobacter pylori, a Gram-negative bacterium, in vitro. PLA2-IIA was not bactericidal against H. pylori, whereas PLA2-XIIA effectively killed H. pylori at a concentration of 50 microg/ml but was not bactericidal at concentrations of 0.5 microg/ml and 5 microg/ml.

Anti-Bacterial Agents↗
PubMed2006

Automated pattern ranking in differential display data analysis.

Gene expression analysis by differential display (DD) is limited by the labor-intensive visual evaluation of the electrophoretic data traces. We describe a flexible method for computer-assisted ranking of expression patterns in data from DD experiments. The method is based on a pairwise alignment and comparison of the quantitative trace data with respect to specific expression patterns defined by the investigator. The observed patterns are ranked according to a score value that identifies the most potential findings to be confirmed visually instead of the vast amount of original results. This two-step approach, enabled by the efficient computer algorithm for gene expression pattern comparison, will increase the percentage of true-positive findings chosen for the tedious downstream processing, while minimizing the cost and labor involved in large scale DD data analysis.

Algorithms↗
PubMed2005

Time-resolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum.

Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A(2) (PLA(2)), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein. Antibodies were raised in rabbits against recombinant human PLA(2) proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 microg/L for the TR-FIA for GIB PLA(2), 1 microg/L (GIIA), 35 microg/L (GIID), 3 microg/L (GIIE), 4 microg/L (GIIF), 14 microg/L (GIII), 11 microg/L (GV), 2 microg/L (GX), 92 microg/L (GXIIA) and 242 microg/L (GXIIB). All secreted PLA(2)s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA(2) in serum (median 19.0 microg/L, range 13.1-33.7 microg/L, n = 4) as compared to the healthy blood donors (median 1.8 microg/L, range 0.8-3.4 microg/L, n = 28, P < 0.0001). The concentration of GIIA PLA(2) in the sera of septic patients (median 315.7 microg/L, range 15.9-979.6 microg/L, n = 34) was highly elevated as compared to that of the blood donors (median 1.8 microg/L, range 0.8-5.8 microg/L, n = 28, P < 0.0001). Our current results confirmed elevated concentrations of GIB and GIIA PLA(2) in the sera of patients suffering from acute pancreatitis or septic infections, respectively, as compared to healthy subjects. However, in the same serum samples, the concentrations of the other secreted PLA(2)s, viz. GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were below the respective analytical sensitivities of the TR-FIAs. It is concluded that generalized bacterial infections do not lead to elevated serum levels of GIIE, GIIF, GIII, GV and GX PLA(2)s above the detection limits of the current TR-FIAs.

Adult↗
PubMed2004

Phospholipase A2 in porifera.

Phospholipase A2 (PLA2) catalytic activity was measured in aqueous extracts of 83 freeze-dried specimens representing 55 marine sponge species collected from the east coast of Australia including the Great Barrier Reef. High levels (>500 u/l) of PLA2 activity (defined as the amount of activity that releases 1 micromol of fatty acid per min) were found in four out of 55 species (7%), moderate activities (100-499 u/l) in 6/55 (11%), low activities (1-99 u/l) in 11/55 (20%) and no PLA2 activity in 34/55 (62%). Species with high PLA2 activity levels included Cymbastela coralliophila (2118 u/l, specific activity 10,590 u/g of protein), Acanthella cavernosa (1318 u/l, specific activity 2470 u/g), Spirastrella vagabunda (1036 u/l, specific activity 1727 u/g and Theonella swinhoei (567 u/l, specific activity 354 u/g). It was postulated that poriferan PLA2 may be involved in eicosanoid metabolism and antimicrobial and toxic defence of the animal.

Animals↗
PubMed2004

Phospholipase A2 in cnidaria.

Phospholipase A2 (PLA2) is an enzyme present in snake and other venoms and body fluids. We measured PLA2 catalytic activity in tissue homogenates of 22 species representing the classes Anthozoa, Hydrozoa, Scyphozoa and Cubozoa of the phylum Cnidaria. High PLA2 levels were found in the hydrozoan fire coral Millepora sp. (median 735 U/g protein) and the stony coral Pocillopora damicornis (693 U/g) that cause skin irritation upon contact. High levels of PLA2 activity were also found in the acontia of the sea anemone Adamsia carciniopados (293 U/g). Acontia are long threads containing nematocysts and are used in defense and aggression by the animal. Tentacles of scyphozoan and cubozoan species had high PLA2 activity levels: those of the multitentacled box jellyfish Chironex fleckeri contained 184 U/g PLA2 activity. The functions of cnidarian PLA2 may include roles in the capture and digestion of prey and defense of the animal. The current observations support the idea that cnidarian PLA2 may participate in the sting site irritation and systemic envenomation syndrome resulting from contact with cnidarians.

Animals↗
PubMed2004

Experimental Helicobacter felis infection in transgenic mice expressing human group IIA phospholipase A2.

BACKGROUND: Both various virulence factors of Helicobacter pylori and host factors influence the clinical outcome of H. pylori infection. In animal experiments with Helicobacter felis, large variations in the severity of disease have been observed between different mouse strains infected with a single isolate of H. felis. C57BL/6 J mouse strain that lacks the expression of group IIA phospholipase A2 has been shown to develop more severe gastric inflammation than other mouse strains. Thus, group IIA phospholipase A2 has been suggested to play a role in regulating inflammation in gastric mucosa. The aim of this study was to examine the possible role of group IIA phospholipase A2 in experimental Helicobacter infection. MATERIALS AND METHODS: Transgenic mice expressing human group IIA phospholipase A2 and their group IIA phospholipase A2 deficient nontransgenic C57BL/6 J littermates were infected with H. felis. The mice were killed 3, 8, and 19 weeks after inoculation of bacteria to determine the histopathological changes in gastric mucosa. RESULTS: The infected mice developed chronic inflammation in gastric mucosa. We found no differences in the colonization of bacteria between transgenic and nontransgenic mice. At 3 and 8 weeks, no difference was found in the severity of inflammation between the two groups. Nineteen weeks after the administration of bacteria the inflammation was more marked in nontransgenic than transgenic mice. Group IIA phospholipase A2 was expressed by in situ hybridization in the neck cells of the glandular stomach in transgenic mice. CONCLUSIONS: The results of the present study suggest that the endogenous expression of group IIA phospholipase A2 diminishes chronic inflammation in gastric mucosa in experimental H. felis infection in mice.

Animals↗
PubMed2003

Diurnal variation in group IIa phospholipase A2 content in tears of contact lens wearers and normal controls.

PURPOSE: To study the diurnal rhythm in group IIA phospholipase A(2) (GIIAPLA(2)) content of tears and the effect of the wearing time of soft contact lenses (CL) on the content of GIIAPLA(2 )in tears. METHODS: The GIIAPLA(2 )content of tears was measured by a time-resolved fluoroimmunoassay in 22 healthy controls at 8 a.m., noon, 4 p.m. and 8 p.m. and in 20 CL wearers at 4 p.m. 1-2 days before using CLs and after 4 h (at noon), 8 h (4 p.m.) and 12 h (8 p.m.) use of soft CLs. RESULTS: The GIIAPLA(2 )content of tears of healthy controls was 80.6+/-47.8 micro g/ml (mean+/-SD). The GIIAPLA(2 )content was lower at 8 a.m. than at noon (p=0.006) and higher at 4 p.m. than at 8 p.m. ( P=0.003). There was no statistically significant difference in the GIIAPLA(2 )content of tears between the CL wearers without CLs (69.47+/-31.2 micro g/ml) and the normal subjects (92.3+/-48.2 micro g/ml) measured at 4 p.m. Compared with healthy controls, the GIIAPLA(2) values in subjects wearing CLs were statistically significantly lower at noon ( P=0.0001) and at 4 p.m. ( P=0.0002). CONCLUSION: In normal subjects, the GIIAPLA(2) content of tears increased from 8 a.m. to noon and decreased from 4 p.m. to 8 p.m. The use of CLs for 4 h and 8 h caused a decrease in the GIIAPLA(2) content of tears. This difference was not seen at 4 p.m. the day when the CL wearers did not use CLs.

Adult↗
PubMed2002

Group IIA phospholipase A2 content of tears in patients with keratoconjunctivitis sicca.

PURPOSE: To determine the concentration of group IIA phospholipase A2 (GIIAPLA(2)) in tears of patients with keratoconjunctivitis sicca (KCS) and to compare it with the GIIAPLA(2) content of tears in age-matched healthy controls. METHODS: The GIIAPLA(2)content of tears was measured with time-resolved fluoroimmunoassay in 20 patients with KCS (mean age 70.7+/-8.7 years) and in 20 normal subjects (mean age 70.2+/-9.9 years). RESULTS: The GIIAPLA(2)content of tears in patients with KCS was 75.8+/-54.2 microg/ml and in normal subjects it was 34.2+/-21.4 microg/ml. The difference was statistically significant. In patients with KCS the GIIAPLA(2) concentration slightly increased with decreasing Schirmer test values (Spearman's correlation =-0.20) CONCLUSION: The results of this study indicate that in patients with KCS the relative concentration of GIIAPLA(2) of tears was increased due to decreased aqueous production in the lacrimal glands and better preservation of the acinar cells in the central parts of the lobules of the lacrimal glands producing GIIAPLA(2).

Aged↗
PubMed2002

Bactericidal group IIA phospholipase A2 in serum of patients with bacterial infections.

Group IIA phospholipase A2 (PLA2-IIA) is a newly recognized antibacterial acute phase protein. The concentration of PLA2-IIA increases up to 500-fold in the blood plasma of patients with severe acute diseases, compared with healthy persons. Despite numerous studies, the exact roles of this enzyme in human diseases are unknown. This study investigated the antibacterial properties of PLA2-IIA in human acute phase serum. PLA2-IIA in serum samples of patients with bacterial infections was capable of killing 90% of Staphylococcus aureus and 99% of Listeria monocytogenes in vitro after incubation for 2 h. At concentrations found in normal human serum, PLA2-IIA killed 90% of L. monocytogenes but did not kill S. aureus or Escherichia coli. The bactericidal effects of acute phase and normal human serum were abolished after depletion of PLA2-IIA by immunoadsorption.

Animals↗
PubMed2002

Catalytic activity of phospholipase A2 in blood plasma and pancreatic tissue perfusion in early experimental acute pancreatitis.

BACKGROUND: To investigate the sequence of changes in the catalytic activity of phospholipase A2 in plasma and pancreatic tissue perfusion and oxygenation in mild and severe acute pancreatitis in pigs. METHODS: Twenty-four pigs were randomized into the groups of severe acute pancreatitis, mild acute pancreatitis, and controls. The pancreatic duct of eight anesthetized and mechanically ventilated pigs was cannulated, and taurocholic acid was infused into the pancreatic duct to induce severe acute pancreatitis. Eight animals received intraductal saline and developed mild acute pancreatitis. Eight pigs were cannulated only and served as controls. RESULTS: Central hemodynamics, arterial blood gases, and acid-base balance were stable throughout the study period in all three groups. Pancreatic tissue oxygenation decreased in pigs with severe acute pancreatitis and increased in animals with mild acute pancreatitis. The catalytic activity of phospholipase A2 in plasma remained stable, and there was no difference between the groups. Similarly, C-reactive protein values remained within the normal range during the study period in all groups. CONCLUSION: Plasma phospholipase A2 levels do not react to the changes in pancreatic tissue perfusion in the early phase of mild and severe acute pancreatitis.

Acute Disease↗
PubMed2002

mRNA differential display of gene expression in colonic carcinoma.

We analyzed changes in gene expression in human colonic carcinoma by fluorescent mRNA differential display. RNA isolated from two samples of normal colon and four cases of colonic adenocarcinoma were amplified with a 15 x 32 set of primers resulting in 2880 cDNAs analyzed with an automated sequencer. Electrophoretic patterns implying constitutive gene expression as well as upregulated and downregulated expression in carcinomas were identified. Forty such cDNA fragments were purified by a novel fluorescent polyacrylamide gel electrophoresis (PAGE)-based method and identified by cyclic sequencing. Most genes showing differential expression were upregulated in colonic carcinoma. Upregulated genes included those for various ribosomal and mitochondrial proteins, heat shock proteins, nucleolar RNA-helicase and phosphoserine aminotransferase. Downregulated genes included histone H3.3. In conclusion, genes associated with vital cellular functions such as transcription, protein synthesis and mitochondrial metabolism were upregulated in colonic carcinoma. Fluorescent mRNA differential display can be applied to the identification of novel cancer-related genes for diagnostic, prognostic and therapeutic purposes.

Adenocarcinoma↗
PubMed2002

Cloning of a novel phospholipase A2 from the cnidarian Adamsia carciniopados.

PLA2 catalytic activity was detected in homogenised tissues, including tentacles and acontia (structures for preying and defence, respectively), of the sea anemone Adamsia carciniopados. Nested reverse transcription polymerase chain reaction (RT PCR) with degenerate primers and rapid amplification of cDNA ends (RACE) were used to clone a novel phospholipase A2 from Adamsia carciniopados (AcPLA2). AcPLA2 contains a putative prepropeptide of 37 residues, ending with a basic doublet followed by a mature protein of 119 amino acids, including 12 cysteines. AcPLA2 displays only 30-42% similarity with other known secretory PLA2s (sPLA2). C-terminal extension, typical of groups II and X PLA2s, is absent. Predicted molecular weight and pI of the mature protein are 13.5 kDa and 9.1, respectively. Structural features and phylogenetic analysis set AcPLA2 apart from the known sPLA2s and define this molecule in the ancient metazoan phylum Cnidaria as a member of a new class of sPLA2s.

Amino Acid Sequence↗
PubMed2002

Group IIA phospholipase A2 content of basal, nonstimulated and reflex tears.

PURPOSE: To determine the concentration of group IIA phospholipase A2 (GIIAPLA(2)) in basal, nonstimulated and reflex tears of young normal subjects. METHODS: The GIIAPLA(2) content of tears was measured in left eye of 16 healthy subjects (mean age 24.5 +/- 2.2 years). Five samples were taken from each subject: nonstimulated sample; basal sample in a dark room after topical anesthesia; and reflex tear samples immediately on stimulation by bright light and breathing of the vapor of onions, and 1 and 3 minutes after the onset of tear flow. RESULTS: The GIIAPLA(2) content of reflex tears was statistically significantly lower than the GIIAPLA(2) content of basal tears, whereas there was no statistically significant difference in the GIIAPLA(2) content between nonstimulated and basal tears. CONCLUSIONS: The current results indicate that the basal and nonstimulated tears are similar in their GIIAPLA(2) content, and that the GIIAPLA( 2) content of tears decreases with stimulation, possibly due to dilution of GIIAPLA(2) content during reflectory hypersecretion of tears.

Adult↗
PubMed2002

Acute pancreatitis in transgenic mice expressing human group IIA phospholipase A2.

INTRODUCTION: There is circumstantial and contradictory evidence of the role of group IIA phospholipase A2 (PLA2) in acute pancreatitis. AIM: To examine the severity of acute experimental pancreatitis in transgenic mice expressing human PLA2 compared with mice not expressing PLA2. METHODS: The study involved 12 young female CB57/bl mice not expressing group IIA PLA2 (wild-type mice) and 12 transgenic female CB57/bl mice expressing human group IIA PLA2 (transgenic mice). A choline-deficient, 0.5% ethionine-supplemented diet induced acute pancreatitis for 72 hours after 12 hours of fasting. Mice were killed 4 and 10 days after induction of acute pancreatitis. Pancreas, lung, kidney, and liver were examined histologically, and apoptosis in pancreas and liver was evaluated by DNA nick-end labeling (TUNEL). RESULTS: On day 4, there were no significant differences in pancreatic apoptosis or total pancreatitis score. Liver damage was similar in both groups. On day 10, pancreatic damage was less but apoptosis more severe than on day 4, and neither hepatic damage nor apoptosis was seen. All mice expressing human group IIA PLA2 but none of the mice not expressing human group IIA PLA2 had marked pancreatic fibrosis. No significant pulmonary or renal damage was found at any time. CONCLUSION: Pancreatitis in mice expressing human group IIA PLA2 is not more severe than in normal mice. Expression of group IIA PLA2 per se is not a major determinant of severity in experimental acute pancreatitis.

Acute Disease↗
PubMed2001

Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A(2).

Group IIA secreted phospholipase A(2) (sPLA2) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of recombinant murine and human groups I, II, V, X, and XII sPLA2s on Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. The rank order potency among human sPLA2s against Gram-positive bacteria is group IIA > X > V > XII > IIE > IB, IIF (for murine sPLA2s: IIA > IID > V > IIE > IIC, X > IB, IIF), and only human group XII displays detectable bactericidal activity against the Gram-negative bacterium E. coli. These studies show that highly basic sPLA2s display potent bactericidal activity with the exception of the ability of the acidic human group X sPLA2 to kill Gram-positive bacteria. By studying the Bacillus subtilis and S. aureus bactericidal potencies of a large panel of human group IIA mutants in which basic residues were mutated to acidic residues, it was found that: 1) the overall positive charge of the sPLA2 is the dominant factor in dictating bactericidal potency; 2) basic residues on the putative membrane binding surface of the sPLA2 are modestly more important for bactericidal activity than are other basic residues; 3) relative bactericidal potency tracks well with the ability of these mutants to degrade phospholipids in the bacterial membrane; and 4) exposure of the bacterial membrane of Gram-positive bacteria by disruption of the cell wall dramatically reduces the negative effect of charge reversal mutagenesis on bactericidal potency.

Amino Acid Substitution↗