Pyroligneous acid from Mimosa tenuiflora and Eucalyptus urograndis as an antimicrobial in dairy goats.

AIMS: To evaluate the applicability of the Mimosa tenuiflora and Eucalyptus urograndis pyroligneous acids (PAs) as alternative antiseptics in dairy goats. METHODS AND RESULTS: Cytotoxicity was evaluated in vitro using bacteria, as well as in vivo using goats, and the influence of PAs on the physicochemical parameters of fresh milk were examined. The cytotoxicity of PAs was evaluated in terms of morphology, cell viability and metabolic activity of goat tegumentary cells. The PA of M. tenuiflora had results similar to those of 2% iodine. For the in vitro tests, strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were used with the well technique, demonstrating inhibition halos greater than 9 mm. In the in vivo test, 15 animals were used per phase of the experiment, and the plate counting technique showed that there was antiseptic action of both extracts, with emphasis on the M. tenuiflora PA. Physicochemical analysis of the milk showed that neither PAs interfered with its physical-chemical parameters. CONCLUSIONS: The PA of M. tenuiflora presented potential as an alternative antiseptic in dairy goats. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the use of PA as an antimicrobial agent in animals.

First molecular evidence of frogs as a food source for sand flies (Diptera: Phlebotominae) in Brazilian caves.

Genus and species of phlebotomine sand flies have been recorded and described in caves in Brazil, but no study has provided the food source used by sand flies in these environments. Herein, we identified the blood source used by sand fly species in caves located at "Quadrilátero Ferrífero" (QF), Minas Gerais state. Specimens were manually collected near or on anurans inside ferruginous caves in Serra do Gandarela National Park and Serra do Rola Moça State Park. Males and females were placed in vials with 70% alcohol and stored at -10°C. Females engorged, after specific identification, had DNA extracted and followed for PCR amplification using specific primers. Sequencing was analyzed in the GenBank and Barcode of Life. A total of 198 specimens were collected (107 females and 91 males), all of them belonging to species Sciopemyia aff. microps (88.89%), Sciopemyia sordellii (10.61%), or Martinsmyia oliveirai (0.50%). When it comes to the females, 89 were S. aff. microps and 18 S. sordellii. Nineteen engorged females of S. aff. microps were analyzed and most of them (n=18) presented blood from Bokermannohyla martinsi and one contained blood from Scinax fuscovarius. The blood present in engorged females of S. sordellii (n=4) was from B. martinsi. Sciopemyia genus specimens are commonly found in collections carried out inside natural caves, but this was the first study to prove that females of this genus feed on cold-blooded animals in nature. HIGHLIGHTS: • Here we proved that sand flies feed in cold-blooded animals in in Brazilian caves. • Females of the Sciopemyia genus were for the first time found feeding in natural habitats. • Anurans of the family Hylidae were identified as source by molecular analyzes. • Insect bloodmeal identification can help assessing the fauna in several biomes. • This is the first record of S. aff. microps in caves of Brazil.

Composition of semen and foreskin mucosa aerobic microbiota and its impact on sperm parameters of captive collared peccaries (Pecari tajacu).

AIM: To evaluate the bacterial composition of collared peccary semen and foreskin mucosa, and to verify the sensitivity of isolates to antimicrobials used in semen conservation and to Aloe vera gel, which is an alternative external cryoprotectant. METHODS AND RESULTS: Nine foreskin mucosa and ejaculate samples from adult animals were used. Sperm characteristics and bacterial load were evaluated in fresh semen. The preputial mucosa and semen bacterial isolates were identified and tested against five concentrations of each antimicrobial (streptomycin-penicillin and gentamicin) and A. vera gel. Corynebacterium sp. and Staphylococcus sp. were isolated in greater numbers than others in both semen (64·10 and 20·51%, respectively) and the foreskin mucosa (60·60 and 24·25%, respectively), and ranged from 0·4 to 21 × 105 colony-forming units (CFU) per ml. The average load of Corynebacterium sp. was negatively correlated (P < 0·05) with the sperm membrane integrity (r = -0·73055) and curvilinear velocity (r = -0·69048). Streptomycin-penicillin and gentamicin inhibited most micro-organisms, and A. vera showed lower antimicrobial activity. CONCLUSION: Several Gram-positive bacteria are present in semen and foreskin mucosa of collared peccary, and the benefits of using primarily penicillin-streptomycin and gentamicin antimicrobials in the bacterial control of diluted semen of these animals are strongly indicated. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insight into the reproductive microbiota of captive male-collared peccary. This work provides a theoretical basis to assist reproductive biotechnologies for ex situ conservation of the species.

Effects of octenidine applied alone or mixed with sodium hypochlorite on eukaryotic cells.

AIM: To assess the effects of octenidine dihydrochloride (OCT) on eukaryotic cells and the cytotoxicity of OCT associated with sodium hypochlorite - NaOCl (NaOCl/OCT). METHODOLOGY: L929 fibroblasts and human osteoblast-like cells (Saos-2) were exposed to 0.1% OCT, 2% CHX, 2.5% NaOCl, 5.25% NaOCl and mixtures of 5.25% NaOCl and 0.1% OCT (NaOCl/OCT) at 90 : 10, 80 : 20 and 50 : 50 ratios. Cell viability was assessed by methyl-thiazol-tetrazolium (MTT) and neutral red (NR) assays; type of cell death, by flow cytometry; cytoskeleton, by actin and α-tubulin fluorescence; and alkaline phosphatase (ALP) activity, by thymolphthalein release. The data were analysed by two-way ANOVA and Bonferroni tests (α = 0.05). RESULTS: MTT and NR assays revealed that 0.1% OCT had the lowest cytotoxicity (P < 0.05), followed by 2% CHX (P < 0.05). The 2.5% NaOCl, NaOCl/OCT 80 : 20 and NaOCl/OCT 50 : 50 solutions had intermediate cytotoxicity. NaOCl 5.25% and NaOCl/OCT 90 : 10 had the highest cytotoxicity (P < 0.05). The OCT group had a higher percentage of viable cells than the NaOCl and CHX groups (P < 0.05), and induced apoptosis at higher doses. The cytoskeleton alterations were observed at 0.12%, 0.6% and 2.02% for the NaOCl, CHX and OCT groups, respectively. The solutions did not induce ALP activity. CONCLUSION: Octenidine dihydrochloride was less cytotoxic, induced apoptosis at higher doses, caused few changes in the cytoskeleton and did not induce alkaline phosphatase activity. In addition, octenidine dihydrochloride reduced the cytotoxicity of 5.25% NaOCl when combined at 20 and 50%.

Distinct beneficial effects of continuous vs accumulated exercise training on cardiovascular risk factors in Wistar rats.

We compared the effects of continuous exercise (CE) vs accumulated exercise (AE) training on CVD risk factors and heart of young male Wistar rats. The exercise training (ET) was performed in a swimming pool for 30-60 min/day, 5 days/week over 15 weeks. CE group performed the ET in a single long daily session (30-60 min), while AE group performed the ET at the same frequency, intensity, and duration of CE rats, but in three short bouts over the course of a day (10-20 min in three daily sessions). AE training was more efficient than CE in attenuating body and fat weight gain and inhibiting visceral adipocyte hypertrophy at the same food intake level. CE training was more efficient in improving systolic blood pressure, LDL/HDL cholesterol, and serum triglyceride. Both ET protocols increased heart function, decreased lipid peroxidation, and increased intracellular Hsp72 content in the heart. This work shows distinct beneficial effects of CE vs AE training suggesting that the prescription of one or other may be preferred to prevent the increase of a specific CVD risk factor.

Diphtheria outbreak in Maranhão, Brazil: microbiological, clinical and epidemiological aspects.

We describe microbiological, clinical and epidemiological aspects of a diphtheria outbreak that occurred in Maranhão, Brazil. The majority of the 27 confirmed cases occurred in partially (n = 16) or completely (n = 10) immunized children (n = 26). Clinical signs and characteristic symptoms of diphtheria such as cervical lymphadenopathy and pseudomembrane formation were absent in 48% and 7% of the cases, respectively. Complications such as paralysis of lower limbs were observed. Three cases resulted in death, two of them in completely immunized children. Microbiological analysis identified the isolates as Corynebacterium diphtheriae biovar intermedius with a predominant PFGE type. Most of them were toxigenic and some showed a decrease in penicillin G susceptibility. In conclusion, diphtheria remains endemic in Brazil. Health professionals need to be aware of the possibility of atypical cases of C. diphtheriae infection, including pharyngitis without pseudomembrane formation.

Brain glucose hypometabolism and hippocampal inflammation in Goto-Kakizaki rats.

Brain glucose hypometabolism and neuroinflammation are early pathogenic manifestations in neurological disorders. Neuroinflammation may also disrupt leptin signaling, an adipokine that centrally regulates appetite and energy balance by acting on the hypothalamus and exerting neuroprotection in the hippocampus. The Goto-Kakizaki (GK) rat is a non-obese type 2 diabetes mellitus (T2DM) animal model used to investigate diabetes-associated molecular mechanisms without obesity jeopardizing effects. Wistar and GK rats received the maintenance adult rodent diet. Also, an additional control group of Wistar rats received a high-fat and high-sugar diet (HFHS) provided by free consumption of condensed milk. All diets and water were provided ad libitum for eight weeks. Brain glucose uptake was evaluated by 2-deoxy-2-[fluorine-18] fluoro-D-glucose under basal (saline administration) or stimulated (CL316,243, a selective ß3-AR agonist) conditions. The animals were fasted for 10-12 h, anesthetized, and euthanized. The brain was quickly dissected, and the hippocampal area was sectioned and stored at -80°C in different tubes for protein and RNA analyses on the same animal. GK rats exhibited attenuated brain glucose uptake compared to Wistar animals and the HFHS group under basal conditions. Also, the hippocampus of GK rats displayed upregulated leptin receptor, IL-1ß, and IL-6 gene expression and IL-1ß and the subunit of the transcription factor NF-κB (p-p65) protein expression. No significant alterations were detected in the hippocampus of HFHS rats. Our data indicated that a genetic predisposition to T2DM has significant brain deteriorating features, including brain glucose hypometabolism, neuroinflammation, and leptin signaling disruption in the hippocampal area.

Serum biomarkers are similar in Churg-Strauss syndrome and hypereosinophilic syndrome.

RATIONALE: Churg-Strauss syndrome (CSS) and hypereosinophilic syndrome (HES) overlap considerably in clinical presentation. A reliable means of distinguishing between these groups of patients is needed, especially in the setting of glucocorticoid therapy. METHODS: A retrospective chart review of 276 adult subjects referred for evaluation of eosinophilia > 1500/µl was performed, and subjects with a documented secondary cause of eosinophilia or a PDGFR -positive myeloproliferative neoplasm were excluded. The remaining subjects were assessed for the presence of American College of Rheumatology (ACR) criteria. Laboratory and clinical parameters were compared between subjects with biopsy-proven vasculitis (CSS; n = 8), ≥4 ACR criteria (probable CSS; n = 21), HES with asthma and/or sinusitis without other CSS-defining criteria (HESwAS; n = 20), HES without asthma or sinusitis (HES; n = 18), and normal controls (n = 8). Serum biomarkers reported to be associated with CSS were measured using standard techniques. RESULTS: There were no differences between the subjects with definite or probable CSS or HES with respect to age, gender, or maintenance steroid dose. Serum CCL17, IL-8, and eotaxin levels were significantly increased in eosinophilic subjects as compared to normal controls, but were similar between the eosinophilic groups. Serum CCL17 correlated with eosinophil count (P < 0.0001, r = 0.73), but not with prednisone dose. CONCLUSIONS: In patients with a history of asthma and sinusitis, distinguishing between ANCA-negative CSS and PDGFR-negative HES is difficult because of significant overlap in clinical presentation and biomarker profiles.

Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains.

During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.

Root deformation affects mineral nutrition but not leaf gas exchange and growth of Genipa americana seedlings during the recovery phase after soil flooding.

Root deformation (RD) caused by errors in the pricking out process are irreversible and very difficult to detect in container-grown seedlings at the time of planting in the field. The objective of this study was to evaluate the effects of RD on leaf gas exchange, growth, biomass allocation and mineral nutrition of G. americana seedlings during the recovery phase after soil flooding. Four-months-old seedlings, with and without RD, were flooded for 42 days and their recovery was evaluated 28 days after soil drainage. There were no significant interactions between RD and soil flooding for all leaf gas exchange, growth and mineral nutrition after soil drainage, with the exception of leaf P concentrations. In plants with no RD, the P concentration in leaves of non-flooded plants was significantly higher than that of plants with RD. Soil flooding and RD did not influence leaf or root N concentrations or whole-plant N content. RD increased the K concentration in the roots, but not in the leaves. Changes in the nutrient concentrations in leaves and roots indicate that RD may affect physiological performance of seedlings after planting in the field.

Microbial source tracking and antimicrobial resistance in one river system of a rural community in Bahia, Brazil.

Use of antibiotics inevitably leads to antimicrobial resistance. Selection for resistance occurs primarily within the gut of humans and animals as well as in the environment through natural resistance and residual antibiotics in streams and soil. We evaluated antimicrobial resistance in Gram negative bacteria from a river system in a rural community in Bahia, Brazil. Water was collected from the Jiquiriçá and Brejões rivers and the piped water supply. Additionally, stools were collected from a random sample of residents, cows, pigs and horses near the river. The samples were screened for bacteria resistant to ciprofloxacin, cefotaxime, and meropenem and identified biochemically at the genus and species levels. Microbial source tracking demonstrated that ruminant and human fecal contamination increased as the rivers neared the village center and decreased after the last residence. Antibiotic bacteria were identified from all samples (n = 32). No bacteria were resistant to carbapenems, but the majority of the enterobacteria were resistant to ciprofloxacin, even though this class of antibiotics is not commonly used in food animals in this region. Considering these facts, together with the pattern of human fecal contamination, a human source was considered most likely for these resistant isolates.

Toxic effects of thallium acetate by acute exposure to the nematode C. elegans.

BACKGROUND: Thallium (Tl) is a toxic metalloid and an emerging pollutant due to electronic devices and dispersal nearby base-metal mining. Therefore, Tl poses a threat to human health and especially the long-term impact on younger individuals exposed is still unknown. This study aimed to evaluate the toxic effects of thallium acetate in C. elegans in early larval stages, considering physiological and behavioral endpoints, as well as the Tl absorption and bioaccumulation. METHODS: Caenorhabditis elegans (C. elegans) was exposed to Thallium acetate (50, 100, 150, 200, 250, 500, and 1000 µM) in the L1 larval stage, with the purpose to observe the toxic effects invoked until adulthood. Transgenic worms strains were transported GFP, reporters to DAF-16 and were used to verify the antioxidant response. ICP-MS quantified total Tl+ concentration to evidence Tl uptake and bioaccumulation. RESULTS: Thallium acetate caused a significant reduction in the number of living worms (p < 0.0001 in 100-1000 µM), a delay in larval development (p < 0.01; p < 0.001 and p < 0.0001 in 100-1000 µM) through the larval stages, and egg production in the worm's uterus was reduced. Thallium acetate also induced behavioral changes. Additionally, thallium acetate activated antioxidant pathway responses in C. elegans by translocating the DAF-16 transcription factor and activation of SOD-3::GFP expression. The Tl+ quantification in worms showed its absorption in the L1 larval stage and bioaccumulation in the body after development. CONCLUSIONS: Thallium acetate reduced survival, delayed development, caused behavioral changes, induced responses inherent to oxidative stress, and serious damage to the worm's reproduction. In addition, C. elegans absorbed and bioaccumulated Tl+. Together, our results highlight the impacts of Tl+ exposure in the early stages of life, even for a short period.

Occlusal and Esthetic Enhancement: A Noninvasive Approach to an Old Dilemma.

This article describes an indirect composite restoration technique for diastema closure and tooth lengthening with a noninvasive approach using regularly available materials such as silicone, composite resin, and an adhesive system. The procedure resulted in occlusal and functional improvement, with diastema closure, protrusive guide adjustment, and an increase in central incisor length. The procedure provided an adequate proportion of the central incisors with an esthetically natural appearance. It also resulted in simple, fast, and accurate manufacturing with a noninvasive esthetic indirect rehabilitation compared with more invasive preparation of ceramic veneers.

Caloric restriction-induced weight loss with a high-fat diet does not fully recover visceral adipose tissue inflammation in previously obese C57BL/6 mice.

Caloric restriction (CR) reduces body weight and systemic inflammation, but the effects on adipose tissue under dietary lipid overload are controversial. We evaluated the effects of CR-induced weight loss with a high-fat diet on adipose tissue inflammation of obese mice. Male mice were assigned into low-fat diet (LF) and high-fat diet (HF) groups. After 8 weeks, the mice in the HF group were reassigned for another 7 weeks into the following 3 conditions: (i) kept in the HF condition; (ii) changed to low-fat diet ad libitum (LFAL); and (iii) changed to high-fat calorie-restricted (RHF) diet to reach LFAL body weight. Serum markers, adipocytokines, morphology, and inflammatory infiltrates in retroperitoneal adipose tissue (RAT) were accessed. The body weights of the LFAL and RHF groups were reduced, equaling the body weights of the LF group. The LFAL mice had restored almost all inflammatory markers as the LF mice, except tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and adiponectin. Compared with the HF group, the RHF group had lowered visceral adiposity, retroperitoneal adipocyte sizes, and RAT inflammatory cell infiltration, as well as TNF-α, interleukin-6, and hepatic and serum C-reactive protein, which were higher than that of the LFAL group; adiponectin and MCP-1 did not change. CR with high-fat diet reduced body weight and attenuated visceral adiposity but did not fully recover visceral tissue inflammation. Novelty Caloric restriction in a high-fat diet ameliorated visceral adiposity. Caloric restriction in a high-fat diet did not recover visceral adipose tissue inflammation.

Genomic analysis of a novel nontoxigenic Corynebacterium diphtheriae strain isolated from a cancer patient.

The genome of a novel nontoxigenic Corynebacterium diphtheriae, strain 5015, isolated from a patient with adenoid cystic carcinoma was sequenced and compared with 117 publically available genomes. This strain is phylogenetically distinct and lacks virulence genes encoding the toxin, BigA and Sdr-like adhesins. Strain 5015 possesses spaD-type and spaH-type pilus gene clusters with a loss of some gene functions, and 31 unique genes that need molecular characterization to understand their potential role in virulence characteristics.

First detection of Corynebacterium ulcerans producing a diphtheria-like toxin in a case of human with pulmonary infection in the Rio de Janeiro metropolitan area, Brazil.

The frequency and severity of human infections associated with Corynebacterium ulcerans appear to be increasing in different countries. Here, we describe the first C. ulcerans strain producing a diphtheria-like toxin isolated from an elderly woman with a fatal pulmonary infection and a history of leg skin ulcers in the Rio de Janeiro metropolitan area.

Hormonal strategy to reduce suckled beef cow handling for timed artificial insemination with sex-sorted semen.

Two experiments were conducted to assess a hormonal strategy developed to reduce animal handling for timed artificial insemination (TAI) with sex-sorted semen. Four-hundred ninety-one (491) suckled beef cows received a progesterone (P4) intravaginal device and 2 mg intramuscular (im) injection of estradiol benzoate (EB) on a randomly chosen day of the estrus cycle (Day 0) in Experiment 1. Cows were treated with 500 µg of sodic cloprostenol (PGF2α) and with 300 IU of eCG at P4 device removal (Day 8); these cows were also randomly assigned to receive 1 mg of estradiol cypionate (EC) administered at P4 device removal (treatment EC-0h) or 1 mg of EB 24 h after P4 device removal (treatment EB-24h). Both treatments were timed inseminated (TAI) with sex-sorted semen 60 h after P4 device removal. Cows treated with EC-0h presented higher pregnancy rate per AI (P/AI) [45.0% (113/251)] than the ones treated with EB-24h [35.4% (85/240); P = 0.03)]. A subset of cows (n = 26) were subjected to ultrasound examination every 12 h after P4 device removal for 96 h in the row in order to determine the time of ovulation. Similar interval between device removal and ovulation was recorded for EB-24h = 70.0 ±â€¯2.9 h vs. EC-0h = 66.0 ±â€¯2.8 h (P = 0.52). Five-hundred ninety-one (591) cows were subjected to the same synchronization protocols and treatments (EC-0h or EB-24h). In addition, they were randomly assigned to a 2 × 2 factorial arrangement aiming at determining the effects of treatment with estradiol (EC-0h vs. EB-24h) and of semen type (Sex-sorted vs. Non-sex-sorted semen). All animals were timed inseminated 60 h after P4 device removal. There was no interaction (P = 0.07) between the ovulation inducer and semen type. The EC protocol led to greater P/AI than EB (P = 0.03). Greater (P = 0.01) P/AI was achieved through treatments with non-sex-sorted semen rather than with sex-sorted semen [sex-sorted (EB-24h = 49.0%; EC-0h = 51.0%) vs. non-sex-sorted semen (EB-24h = 52.4%; EC-0h = 68.2%)]. Therefore, EC administered at P4 device removal resulted in greater P/AI. Furthermore, the EC-0h protocol allowed reducing suckled beef cow handing for timed artificial insemination with sex-sorted semen.

Brain glucose hypometabolism and hippocampal inflammation in Goto-Kakizaki rats

Brain glucose hypometabolism and neuroinflammation are early pathogenic manifestations in neurological disorders. Neuroinflammation may also disrupt leptin signaling, an adipokine that centrally regulates appetite and energy balance by acting on the hypothalamus and exerting neuroprotection in the hippocampus. The Goto-Kakizaki (GK) rat is a non-obese type 2 diabetes mellitus (T2DM) animal model used to investigate diabetes-associated molecular mechanisms without obesity jeopardizing effects. Wistar and GK rats received the maintenance adult rodent diet. Also, an additional control group of Wistar rats received a high-fat and high-sugar diet (HFHS) provided by free consumption of condensed milk. All diets and water were provided ad libitum for eight weeks. Brain glucose uptake was evaluated by 2-deoxy-2-[fluorine-18] fluoro-D-glucose under basal (saline administration) or stimulated (CL316,243, a selective β3-AR agonist) conditions. The animals were fasted for 10-12 h, anesthetized, and euthanized. The brain was quickly dissected, and the hippocampal area was sectioned and stored at -80°C in different tubes for protein and RNA analyses on the same animal. GK rats exhibited attenuated brain glucose uptake compared to Wistar animals and the HFHS group under basal conditions. Also, the hippocampus of GK rats displayed upregulated leptin receptor, IL-1β, and IL-6 gene expression and IL-1β and the subunit of the transcription factor NF-κB (p-p65) protein expression. No significant alterations were detected in the hippocampus of HFHS rats. Our data indicated that a genetic predisposition to T2DM has significant brain deteriorating features, including brain glucose hypometabolism, neuroinflammation, and leptin signaling disruption in the hippocampal area.

Nuclei contain two differentially regulated pools of diacylglycerol.

A number of recent studies have highlighted the presence of a nuclear pool of inositol lipids [1] [2] that is regulated during progression through the cell cycle [1] [3], differentiation [1] [2] and after DNA damage [2], suggesting that a number of different regulatory pathways impinge upon this pool of lipids. It has been suggested that the downstream consequence of the activation of one of these nuclear phosphoinositide (PI) regulatory pathways is the generation of nuclear diacylglycerol (DAG) [1] [3] [4], which is important in the activation of nuclear protein kinase C (PKC) [5] [6] [7]. Activation of PKC in turn appears to regulate the progression of cells through G1 and into S phase [4] and through G2 to mitosis [3] [8] [9] [10] [11]. Although the evidence is enticing, there is as yet no direct demonstration that nuclear PIs can be hydrolysed to generate nuclear DAG. Previous data in murine erythroleukemia (MEL) cells have suggested that nuclear phosphoinositidase Cbeta1 (PIC-beta1) activity is important in the generation of nuclear DAG. Here, we demonstrate that the molecular species of nuclear DAG bears little resemblance to the PI pool and is unlikely to be generated directly by hydrolysis of these inositol lipids. Further, we show that there are in fact two distinct subnuclear pools of DAG; one that is highly disaturated and mono-unsaturated (representing more than 90% of the total nuclear DAG) and one that is highly polyunsaturated and is likely to be derived from the hydrolysis of PI. Analysis of these pools, either after differentiation or during cell-cycle progression, suggests that the pools are independently regulated, possibly by the regulation of two different nuclear phospholipase Cs (PLCs).

A novel pathway of cellular phosphatidylinositol(3,4,5)-trisphosphate synthesis is regulated by oxidative stress.

BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is a key second messenger found ubiquitously in higher eukaryotic cells. The activation of Class I phosphoinositide 3-kinases and the subsequent production of PtdIns(3,4,5)P(3) is an important cell signaling event that has been causally linked to the activation of a variety of downstream cellular processes, such as cell migration and proliferation. Although numerous proteins regulating a variety of biological pathways have been shown to bind PtdIns(3,4,5)P(3), there are no data to demonstrate multiple mechanisms for PtdIns(3,4,5)P(3) synthesis in vivo. RESULTS: In this study, we demonstrate an alternative pathway for the in vivo production of PtdIns(3,4,5)P(3) mediated by the action of murine Type Ialpha phosphatidylinositol 4-phosphate 5-kinase (Type Ialpha PIPkinase), an enzyme best characterized as regulating cellular PtdIns(4,5)P(2) levels. Analysis of this novel pathway of PtdIns(3,4,5)P(3) synthesis in cellular membranes leads us to conclude that in vivo, Type Ialpha PIPkinase also acts as a PtdIns(3,4)P(2) 5-kinase. We demonstrate for the first time that cells actually contain an endogenous PtdIns(3,4)P(2) 5-kinase, and that during oxidative stress, this enzyme is responsible for PtdIns(3,4,5)P(3) synthesis. Furthermore, we demonstrate that by upregulating the H(2)O(2)-induced PtdIns(3,4,5)P(3) levels using overexpression studies, the endogenous PtdIns(3,4)P(2) 5-kinase is likely to be Type Ialpha PIPkinase. CONCLUSIONS: We describe for the first time a novel in vivo activity for Type Ialpha PIPkinase, and a novel pathway for the in vivo synthesis of functional PtdIns(3,4,5)P(3), a key lipid second messenger regulating a number of diverse cellular processes.