Aging-associated DNA methylation changes in middle-aged individuals: the Young Finns study.

BACKGROUND: Chronological aging-associated changes in the human DNA methylome have been studied by multiple epigenome-wide association studies (EWASs). Certain CpG sites have been identified as aging-associated in multiple studies, and the majority of the sites identified in various studies show common features regarding location and direction of the methylation change. However, as a whole, the sets of aging-associated CpGs identified in different studies, even with similar tissues and age ranges, show only limited overlap. In this study, we further explore and characterize CpG sites that show close relationship between their DNA methylation level and chronological age during adulthood and which bear the relationship regardless of blood cell type heterogeneity. RESULTS: In this study, with a multivariable regression model adjusted for cell type heterogeneity, we identified 1202 aging-associated CpG sites (a-CpGs, FDR < 5%), in whole blood in a population with an especially narrow age range (40 - 49 years). Repeatedly reported a-CpGs located in genes ELOVL2, FHL2, PENK and KLF14 were also identified. Regions with aging-associated hypermethylation were enriched regarding several gene ontology (GO) terms (especially in the cluster of developmental processes), whereas hypomethylated sites showed no enrichment. The genes with higher numbers of a-CpG hits were more often hypermethylated with advancing age. The comparison analysis revealed that of the 1202 a-CpGs identified in the present study, 987 were identified as differentially methylated also between nonagenarians and young adults in a previous study (The Vitality 90+ study), and importantly, the directions of changes were identical in the previous and in the present study. CONCLUSIONS: Here we report that aging-associated DNA methylation features can be identified in a middle-aged population with an age range of only 9 years. A great majority of these sites have been previously reported as aging-associated in a population aged 19 to 90 years. Aging is associated with different types of changes in DNA methylation, clock-like as well as random. We speculate that the a-CpGs identified here in a population with a narrow age-range represent clock-like changes, as they showed concordant methylation behavior in population spanning whole adulthood as well.

Effect of procainhydrochloride on phospholipase A2 catalytic activity in sodium taurocholate-induced acute experimental pancreatitis in rats.

BACKGROUND: In human acute pancreatitis (AP) the local anaesthetic procainhydrochloride (procain-HCl) is given intravenously for pain treatment. Procain has been shown to inhibit catalytic activity of pancreatic (group I) phospholipase A2 (PLA2) and non-pancreatic (group II) PLA2. Both enzymes are important mediators for the local and systemic inflammatory process in AP. To determine the effect of procain, we examined serum and tissue levels of both types of PLA2 activity in the experimental rodent taurocholate model of AP. METHODS: In 60 rats, severe pancreatitis was induced by taurocholate. Forty rats were treated with procain-HCl intravenously at a dosage of 2 mg/kg body weight/h either at or 1 h after induction of pancreatitis. Twenty rats served as controls. We measured catalytic activities of group I and group II PLA2 in serum and tissue samples of lung and pancreas. RESULTS: Serum group II PLA2 catalytic activity was significantly reduced 3 and 6 h after AP induction in rats treated with procain-HCl (p < 0.001) in both treatment groups. In pancreatic and lung tissue, group II PLA2 catalytic activity was significantly reduced compared with normal values (p < 0.001). CONCLUSION: Procain-HCl given intravenously either at or 1 h after induction of necrotizing pancreatitis significantly inhibits group II PLA2 catalytic activity in serum and tissues.

A novel dietary restriction method for group-housed rats: weight gain and clinical chemistry characterization.

Laboratory rodents are usually fed ad libitum. Moderate dietary restriction decreases mortality and morbidity compared with ad libitum feeding. There are, however, problems in achieving dietary restriction. Traditional methods of restricted feeding may interfere with the diurnal rhythms of the animals and are not compatible with group-housing of rodents. We have invented a novel method, the diet board, for restricting the feed intake of laboratory rats. The use of the diet board moderately decreased weight gain of rats when compared with ad libitum-fed animals. The diet board retarded skeletal growth only minimally, whereas major differences were found in body fat depositions. Serum free fatty acid, triglyceride and cholesterol values were lower in diet-restricted rats, while the opposite was true for serum creatine kinase. There were no differences in total protein, albumin or alanine aminotransferase. Moreover, differences in interindividual variances in parameters were not detected between the groups; hence this study could not combine the diet board with reduction potential. The diet board provides mild to moderate dietary restriction for group-housed rats and is unlikely to interfere with the diurnal eating rhythm. The diet board can also be seen as a cage furniture item, dividing the open cage space and increasing the structural complexity of the environment. In conclusion, the diet board appears to possess refinement potential when compared with traditional methods of dietary restriction.

The diet board: welfare impacts of a novel method of dietary restriction in laboratory rats.

Laboratory rats are commonly fed ad libitum (AL). Moderate dietary restriction (DR) decreases mortality and morbidity when compared with AL feeding, but there are several obstacles to the implementation of DR. Traditional methods of restricted feeding disrupt normal diurnal eating rhythms and are not compatible with group housing. We have designed a novel method, the diet board, to restrict the feeding of group-housed rats. Animals fed from the diet board had 15% lower body weight than the AL-fed animals at the age of 17 weeks. The welfare effects of diet board feeding were assessed by comparing the stress physiology of diet board fed animals with that of AL-fed animals. Diet board feeding was associated with higher serum corticosterone levels and lower faecal secretion of IgA, suggesting the diet board causes a stress reaction. However, the AL-fed group had larger adrenal glands with higher adrenaline and noradrenaline content than the diet board animals. No gastric ulcers were found in any of the animals at necropsy. The diet board thus appears to cause a stress reaction when compared with AL-fed rats, but no apparent pathology was associated with this reaction. The diet board could help to solve the health problems associated with AL feeding, while allowing the rats to be group-housed and to maintain their normal diurnal eating rhythms. The diet board can also be seen as a functional cage furniture item, dividing the cage into compartments and thus increasing the structural complexity of the environment. In conclusion, the diet board appears to possess refinement potential compared with traditional methods of DR.

Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.

Group II PLA2 has been implicated in inflammatory processes in both man and other animals and has been shown to be involved in inflammatory conditions, such as arthritis and sepsis. Transgenic mice expressing the human group II PLA2 gene have been generated using a 6.2-kb genomic fragment. These mice express the group II PLA2 gene abundantly in liver, lung, kidney, and skin, and have serum PLA2 activity levels approximately eightfold higher than nontransgenic littermates. The group II PLA2 transgenic mice reported here exhibit epidermal and adnexal hyperplasia, hyperkeratosis, and almost total alopecia. The chronic epidermal hyperplasia and hyperkeratosis seen in these mice is similar to that seen in a variety of dermatopathies, including psoriasis. However, unlike what is seen with these dermatopathies, no significant inflammatory-cell influx was observed in the skin of these animals, or in any other tissue examined. These mice provide an important tool for examining group II PLA2 expression, and for determining the role of group II PLA2 in normal and disease physiology. They serve as an in vivo model for identifying inhibitors of group II PLA2 activity and gene expression.

Pair housing of rabbits reduces variances in growth rates and serum alkaline phosphatase levels.

New Council of Europe regulations mandate housing of two rabbits in the same cage space currently used to house one, provided the animals are socially compatible. This study was designed to assess changes in growth and selected serum chemistry parameters due to pair housing or single housing of rabbits. Six sets of four female siblings of Crl:KBL(NZW)BR rabbits were used. The animals were seven weeks old on arrival. Two siblings of each set were allocated to pair housing, two to single housing. The animals were housed in stainless steel cages (120 cm x 60 cm x 60 cm) with a perforated floor, including a shelf (60 cm x 30 cm) at 30 cm height from the floor. The rabbits were provided with an aspen cube (5 cm x 5 cm x 5 cm), one item per animal. The rabbits were weighed and blood samples were taken from the auricular central artery at four different times during the study. Blood sera were assayed for a set of routinely assayed clinical chemistry parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (APHOS), blood urea nitrogen (BUN), cholesterol (CHOL) and protein (PROT). Mean and variance profiles over the study period were statistically analysed by multivariate analysis of variance. No differences in mean profiles were detected; however, weight (P = 0.0002) and APHOS (P = 0.017) variances were significantly lower in pair-housed animals. The reduction in variance on growth and APHOS attributable to pair housing appears to be rather large. During the 21-week study, occasional fighting was seen between the pair-housed rabbits. After sexual maturity, further major fighting bouts resulted in significant trauma that necessitated the cessation of the study. In conclusion, pair housing appears to have a decreasing effect on growth and APHOS variance, but antisocial behaviour such as fighting remains a serious problem.

CB2 receptor activation causes an ERK1/2-dependent inflammatory response in human RPE cells.

A chronic low-level inflammation contributes to the pathogenesis of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in Western countries. The loss of central vision results from attenuated maintenance of photoreceptors due to the degeneration of retinal pigment epithelium (RPE) cells beneath the photoreceptor layer. It has been proposed that pathologic inflammation initiated in RPE cells could be regulated by the activation of type 2 cannabinoid receptors (CB2). Here, we have analysed the effect of CB2 activation on cellular survival and inflammation in human RPE cells. RPE cells were treated with the selective CB2 agonist JWH-133 in the presence or absence of the oxidative stressor 4-hydroxynonenal. Thereafter, cellular viability as well as the release of pro-inflammatory cytokines and potential underlying signalling pathways were analysed. Our results show that JWH-133 led to increased intracellular Ca2+ levels, suggesting that RPE cells are capable of responding to a CB2 agonist. JWH-133 could not prevent oxidative stress-induced cell death. Instead, 10 µM JWH-133 increased cell death and the release of proinflammatory cytokines in an ERK1/2-dependent manner. In contrast to previous findings, CB2 activation increased, rather than reduced inflammation in RPE cells.

Role of cage material, working style and hearing sensitivity in perception of animal care noise.

During daily care, laboratory animals are exposed to a variety of sounds which may have effects on welfare and also cause physiological and behavioural changes. So far, almost no attention has been paid to individual sounds or the sound level caused by animal care or the sound level inside the animal cage. In this study, sounds from selected rat care procedures were recorded: pulling cage out of the rack, placing it onto a table and replacing the cage back into the rack; with measurements made inside the rat cage and in the adjacent cage. Diet was poured into the food hopper and sounds were recorded inside the cage and also the adjacent cage. The work was repeated in a calm and also in a hurried style, using stainless steel and polycarbonate cages. Finally, the sounds produced by running tap water were recorded. Differences between rat and human hearing were compared using novel species-specific sound level weightings: R-weighting for rats dB(R) and H-weighting for human dB(H). Hurried work with steel caused sound exposure levels exceeding 90 dB(R) when the cages were placed into the rack and about 80 dB(R) when pulling them out of the rack or placing onto a table. With polycarbonate, the levels were 10-15 dB(R) lower. Unhurried calm working produced lower sound exposure levels than hurried working in many procedures. When the procedures were repeated with measurements in the adjacent cage, the sound exposure levels were lower, but the results were similar. Pouring food pellets into a hopper above the rat's head caused 15 dB(R) higher sound exposure levels than pouring food to an adjacent cage. In general, humans hear these sounds about 10-15 dB louder than rats. In conclusion, cage material, working style and hearing sensitivity all have an impact on the sound exposure level in the rodent cage. With correct working methods, high sound levels can be efficiently avoided in most cases.

The trajectory of the blood DNA methylome ageing rate is largely set before adulthood: evidence from two longitudinal studies.

The epigenetic clock, defined as the DNA methylome age (DNAmAge), is a candidate biomarker of ageing. In this study, we aimed to characterize the behaviour of this marker during the human lifespan in more detail using two follow-up cohorts (the Young Finns study, calendar age i.e. cAge range at baseline 15-24 years, 25-year-follow-up, N = 183; The Vitality 90+ study, cAge range at baseline 19-90 years, 4-year-follow-up, N = 48). We also aimed to assess the relationship between DNAmAge estimate and the blood cell distributions, as both of these measures are known to change as a function of age. The subjects' DNAmAges were determined using Horvath's calculator of epigenetic cAge. The estimate of the DNA methylome age acceleration (Δ-cAge-DNAmAge) demonstrated remarkable stability in both cohorts: the individual rank orders of the DNAmAges remained largely unchanged during the follow-ups. The blood cell distributions also demonstrated significant intra-individual correlation between the baseline and follow-up time points. Interestingly, the immunosenescence-associated features (CD8+CD28- and CD4+CD28- cell proportions and the CD4/CD8 cell ratio) were tightly associated with the estimate of the DNA methylome age. In summary, our data demonstrate that the general level of Δ-cAge-DNAmAge is fixed before adulthood and appears to be quite stationary thereafter, even in the oldest-old ages. Moreover, the blood DNAmAge estimate seems to be tightly associated with ageing-associated shifts in blood cell composition, especially with those that are the hallmarks of immunosenescence. Overall, these observations contribute to the understanding of the longitudinal aspects of the DNAmAge estimate.

Roles of secretory phospholipases A(2) in inflammatory diseases and trauma.

Six distinct secretory small molecular weight phospholipases A(2) (PLA(2)) have been cloned and characterized from human tissues. Two of them, pancreatic group IB PLA(2) (PLA(2)-IB) and synovial-type group IIA PLA(2) (PLA(2)-IIA) have been studied as to their association to various inflammatory diseases. PLA(2)-IB is a digestive enzyme synthesized by pancreatic acinar cells. In acute pancreatitis, which is characterized by destruction of pancreatic tissue, PLA(2)-IB is released into the circulation, but its role in pancreatic and other tissue damage is still hypothetical. The concentration of PLA(2)-IIA increases in blood plasma in generalized inflammatory response resulting from infections, chronic inflammatory diseases, acute pancreatitis, trauma and surgical operations. PLA(2)-IIA is synthesized in a number of gland cells and is present in cellular secretions on mucosal surfaces including Paneth cells of intestinal mucosa, prostatic gland cells and seminal plasma, and lacrimal glands and tears. PLA(2)-IIA is expressed in hepatoma-derived cells in vitro and hepatocytes in vivo. PLA(2)-IIA is regarded as an acute phase protein and seems to function as an antibacterial agent especially effective against Gram-positive bacteria. Other putative functions in the inflammatory reaction include hydrolysis of cell membrane phospholipids and release of arachidonic acid for prostanoid synthesis.

Targeting the cannabinoid CB2 receptor: mutations, modeling and development of CB2 selective ligands.

Since the discovery of the cannabinoid CB2 receptor in 1993, there has been a growing interest to clarify the importance of this G-protein coupled receptor (GPCR) for human physiology, and to investigate it as a possible target for current and future drug development. Several mutation studies have examined the receptor activation and structure of the receptor binding cavity. Additionally, 3D models for the CB2 receptor have been constructed to aid in perceiving important ligand-receptor interactions. In recent years, many research groups have succeeded in synthesizing new CB2 selective ligands. This review focuses on (i) important features for ligand recognition and/or receptor activation at CB2, derived from mutation and modeling studies, and (ii) recent advances in the field of CB2 selective ligands.

Anaerobic glycolysis and the development of ischaemic contracture in isolated rat heart.

The relationship between myocardial ATP content and the increase in left ventricular resting tension during ischaemia (ischaemic contracture) was studied in isolated rat hearts perfused for 15 min either with aerobic buffer (pO2 greater than 500 mmHg) containing non-glycolytic substrate, acetate (5 mmol X litre-1), or with hypoxic buffer (pO2 less than 10 mmHg) with glucose (10 mmol X litre-1) before making them totally ischaemic for 10 min. ATP was determined spectrophotometrically from extracts of frozen whole hearts. Left ventricular tension was recorded by intraventricular balloon catheter. Myocardial ATP content was 15.4 +/- 1.0 mumol X g-1 dry weight (mumol X g-1) after 10 min stabilising period, 14.1 +/- 0.9 mumol X g-1 after 15 min aerobic perfusion (plus acetate) and 9.0 +/- 1.3 mumol X g-1 after 15 min hypoxic perfusion (plus glucose). During 10 min of ischaemia ATP decreased in aerobic hearts to 5.4 +/- 1.1 mumol X g-1 and to 7.9 +/- 1.0 mumol X g-1 in hypoxic hearts. The left ventricular resting tension increased during ischaemia in hypoxic hearts to 9 +/- 5% of control systolic pressure (0 = diastolic pressure, 100 = systolic pressure during stabilising period), whereas in aerobic hearts the tension began to increase immediately and was 84 +/- 22% of systolic pressure at the end of the ischaemic period. In parallel control experiments, hearts were also perfused either with glucose-containing aerobic buffer or acetate-containing hypoxic buffer. ATP was well preserved during aerobic perfusion (plus glucose) but decreased markedly during hypoxic perfusion (plus acetate). There was no increase in resting tension in the aerobic hearts (plus glucose) whereas the resting tension increased considerably during hypoxic perfusion (plus acetate). The results indicate that the initiation of ischaemic contracture occurs at much higher myocardial ATP level when ATP comes from mitochondrial sources than when ATP is generated by anaerobic glycolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptomic and epigenetic analyses reveal a gender difference in aging-associated inflammation: the Vitality 90+ study.

Aging is associated with a pro-inflammatory state, often referred to as inflammaging. The origin of the pro-inflammatory mediators and their role in the pathogenesis of the aging-associated diseases remain poorly understood. As aging is also associated with profound changes in the transcriptomic and epigenetic (e.g., DNA methylation) profiles of cells in the peripheral blood, we analyzed the correlation of these profiles with inflammaging using the "classical" marker interleukin-6 as an indicator. The analysis of the whole-genome peripheral blood mononuclear cell (PBMC) gene expression revealed 62 transcripts with expression levels that significantly correlated with the plasma interleukin-6 (IL-6) levels in men, whereas no correlations were observed in women. The Gene Ontology analysis of plasma IL-6-associated transcripts in men revealed processes that were linked to the inflammatory response. Additionally, an Ingenuity Pathway Analysis (IPA) pathway analysis identified Tec kinase signaling as an affected pathway and upstream regulator analysis predicted the activation of IL-10 transcript. DNA methylation was assessed using a HumanMethylation450 array. Seven genes with expression profiles that were associated with the plasma IL-6 levels in men were found to harbor CpG sites with methylation levels that were also associated with the IL-6 levels. Among these genes were IL1RN, CREB5, and FAIM3, which mapped to a network of inflammatory response genes. According to our results, inflammaging is manifested differently at the genomic level in nonagenarian men and women. Part of this difference seems to be of epigenetic origin. These differences point to the genomic regulation of inflammatory response and suggest that the gender-specific immune system dimorphism in older individuals could be accounted for, in part, by DNA methylation.

Increased serum phospholipase A2 activity in schizophrenia: a replication study.

Phospholipase A2 (PLA2) is a key enzyme in the metabolism of phospholipids. Because a disordered phospholipid metabolism has frequently been reported in schizophrenia, we investigated the PLA2 activity in serum from 14 drug-free paranoid schizophrenic patients, 20 healthy controls, and 8 nonschizophrenic psychiatric patients. Schizophrenics showed significantly higher PLA2 activity than healthy controls and nonschizophrenic patients. The increment in schizophrenics was not due to increased concentration of pancreatic secretory PLA2, as concerning pancreatic PLA2 no differences were found among the 3 proband groups. The present findings confirm the results of our previous study and suggest that increased serum PLA2 activity might reflect an increment in the intracellular enzyme activity in schizophrenia. In the brain the activation of intracellular PLA2 results in changes in neuronal activity due to alterations in receptor sensitivity and in neurotransmitter metabolism. The possibility that such PLA2-induced mechanisms are involved in the pathogenesis of schizophrenia should be investigated in further experiments.

Effect of clodronate on fracture healing in denervated rats.

The effect of clodronate on healing of the fracture of osteopenic bone was studied in rats. A total of 165 female rats (14 +/- 1 weeks, 216 +/- 2 g) were divided into five fracture groups (n = 30), and a neurectomized group (n = 15). Osteopenia (op) was induced by right sciatic neurectomy 4 weeks before the fracture. Nonosteopenic (nop) rats were not operated. A closed prepinned diaphyseal fracture of the right femur was done by three-point bending method both to op and nop rats, and the left femur served as an unoperated control. All the fracture groups were divided into treatment (clodronate 10 mg/kg/day sc) and control (saline sc) groups, and the administration was continued throughout the study. The op rats were killed 2, 4, 8, and 12 weeks and nop rats 8 weeks after the fracture. Fracture healing was examined by x-ray and bone-bending strength. Neurectomy reduced bone strength (p < 0.01) at 4 weeks. Clodronate did not affect the bending strength of healing callus of op rats at 2, 4, 8, or 12 weeks after fracture, but reduced the strength of healing callus in nop rats (p < 0.05) at 8 weeks. Radiologic callus width increased in clodronate-treated groups both in op (8 and 12 weeks, p < 0.001) and nop rats (8 weeks, p < 0.05) when compared with saline-treated groups. Clodronate did not affect normal bone strength. In conclusion, clodronate did not affect the bending strength of op fracture nor the strength of the control bones. The remodeling of the fracture was delayed with clodronate.

Synthesis and in vitro evaluation of novel morpholinyl- and methylpiperazinylacyloxyalkyl prodrugs of 2-(6-methoxy-2-naphthyl)propionic acid (Naproxen) for topical drug delivery.

Various novel morpholinyl- (3a,b) and methylpiperazinylacyloxyalkyl (3c-f) esters of 2-(6-methoxy-2-naphthyl)propionic acid were synthesized and evaluated in vitro for topical drug delivery as potential prodrugs of naproxen (1). Compounds 3a-f were prepared by coupling the corresponding naproxen hydroxyalkyl ester with the morpholinyl- or (4-methyl-1-piperazinyl)acyl acid in the presence of dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP) and quantitatively hydrolyzed (t(1/2) = 1-26 min) to naproxen in human serum. Compounds 3c-f showed higher aqueous solubility and similar lipophilicity, determined by their octanol-buffer partition coefficients (log P(app)), at pH 5.0 when compared to naproxen. At pH 7.4 they were significantly more lipophilic than naproxen. The best prodrug 3c led to a 4- and 1.5-fold enhancement of skin permeation when compared to naproxen at pH 7.4 and 5.0, respectively. The present study indicates using a methylpiperazinyl group yields prodrugs that are partially un-ionized under neutral and slightly acidic conditions, and thus, a desirable combination is achieved in terms of aqueous solubility and lipophilicity. Moreover, the resulting combination of biphasic solubility and fast enzymatic hydrolysis of the methylpiperazinylacyloxyalkyl derivatives gave improved topical delivery of naproxen.

Electrophoretic signal comparison applied to mRNA differential display analysis.

Gene expression analysis by electrophoretic methods is currently limited by the labor-intensive visual evaluation of the electrophoretic signal profiles. For this purpose, we present a flexible approach to computer-assisted comparison of quantitative electrophoretic patterns between multiple expression signals. Gaussian curves are first fitted to the complex peak mixtures, and the resulting approximate signals are then aligned and compared on a peak-by-peak basis with respect to specific patterns defined by the investigator. The rationale of the method is to produce a compressed list of exceptional expression patterns quantified by a set of associated numeric features. A score value is attached to each pattern in such a way that large values identify the most potential findings to be focused on in visual analysis instead of the vast amount of original electrophoretic results. The validity of the method is demonstrated by analyzing a large set of electrophoretic data from mRNA differential display experiments monitoring changes in gene expression patterns in human colonic carcinoma. The automated identification of variously defined gene expression patterns agrees well with the visual evaluation of the same electropherograms. The general comparison approach may also be found useful with other gene expression profiling instruments.

Expression of human group II phospholipase A2 in transgenic mice.

Group II phospholipase A2 (PLA2) has been proposed to play an important role in inflammation and defense against bacterial infection. We investigated tissues of transgenic mice expressing the human group II PLA2 gene by immunohistochemistry using rabbit anti-human group II PLA2 antibodies, and by in situ hybridization by probing with human group II PLA2 mRNA anti-sense (test) and sense (control) riboprobes. By immunohistochemistry, human group II PLA2 was found in various mouse tissues and cell types including hepatocytes, proximal tubule cells of the kidney, epithelial cells of the renal pelvis, urinary bladder and ureter, granulosa cells of Graafian follicles, aortic intima and media, cartilage, epiphyseal bone, bronchial epithelial cells, and connective tissue cells in the dermis. By in situ hybridization, group II PLA2 mRNA was localized in hepatocytes, epidermal cells, dermal cells, connective tissue fibroblasts, epithelial and smooth muscle cells of the urinary bladder, and cells of Bowman's capsule. These results show that human group II PLA2 is expressed in large amounts in hepatocytes and many extrahepatic tissues of the transgenic mice. These animals provide a useful new tool for studies on the metabolism, in vivo effects, and physiological and pathological roles of phospholipase A2.

Distribution of group II phospholipase A2 protein and mRNA in rat tissues.

Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.

Secretion of group 2 phospholipase A2 by lacrimal glands.

PURPOSE: Tears are known to have antimicrobial properties. The authors investigated the presence of the antibacterial enzyme phospholipase A2 (PLA2) in tears and lacrimal glands. METHODS: The catalytic activity of PLA2 and the amount of pancreatic group 1 PLA2 and nonpancreatic group 2 PLA2 were measured in homogenates of eight human lacrimal glands from autopsied subjects and in tears from four healthy volunteers. The localization of PLA2s in lacrimal gland sections was studied by immunohistochemistry. Skeletal muscle was used as a control. RESULTS: The catalytic activity of PLA2 was significantly higher in lacrimal glands than in skeletal muscle. Immunochemical analysis showed significantly higher amounts of group 2 PLA2 in lacrimal gland than in skeletal muscle homogenates. Group 1 PLA2 was present in trace amounts only. The concentration of group 2 PLA2 in tears was high (1451.3 micrograms/l) compared to that in the serum of healthy individuals (3.7 micrograms/l). By immunohistochemistry, a granular reaction of group 2 PLA2 was localized in the glandular cells of lacrimal glands. The apical cytoplasm of many duct cells also was labeled. CONCLUSIONS: The lacrimal glands secreted nonpancreatic group 2 PLA2, which most likely acts as an antiinfectious factor in tears.