Altered brain iron deposition in patients with minimal hepatic encephalopathy: an MRI quantitative susceptibility mapping study.

AIM: To explore the use of quantitative susceptibility mapping (QSM) in assessing changes in brain iron deposits and their association with cognitive function in patients with minimal hepatic encephalopathy (MHE). MATERIALS AND METHODS: The study cohort comprised 27 cases with hepatitis B-associated cirrhosis with MHE (MHE group), 25 with hepatitis B-associated cirrhosis without MHE (NMHE group), and 25 healthy controls (HC group). Iron deposits in the bilateral frontal white matter, caudate nucleus (CN), putamen, globus pallidus, thalamus, red nucleus, substantia nigra (SN), hippocampus, and dentate nucleus were measured by QSM. The associations between iron deposition with the time taken to complete number connection tests A (NCT-A) and the score on digital-symbol test (DST) were analysed. RESULTS: Susceptibility values differed significantly in the bilateral CN, left thalamus, right SN, and left hippocampus in the MHE group compared with the other groups and were positively associated with the times taken to complete the NCT-A in the bilateral CN, left thalamus, and right SN and negatively associated with DST scores in the bilateral CN, left TH, and left HP. CONCLUSION: Reduced cognitive function in MHE patients was significantly associated with abnormally increased iron deposition in certain brain areas. The quantification of brain iron deposition by QSM may thus be an objective and accurate means of evaluating MHE.

What physicians do in case of a failure of the pace-sense part of a defibrillation lead : Survey in Germany, Austria and Switzerland.

BACKGROUND: The possible treatment strategies for defects of the pace-sense (P/S) part of a defibrillation lead are either implantation of a new high-voltage (HV)-P/S lead, with or without extraction of the malfunctioning lead, or implantation of a P/S lead. METHODS: We conducted a Web-based survey across cardiac implantable electronic device (CIED) centers to investigate their procedural practice and decision-making process in cases of failure of the P/S portion of defibrillation leads. In particular, we focused on the question of whether the integrity of the HV circuit is confirmed by a test shock before decision-making. The questionnaire included 14 questions and was sent to 951 German, 341 Austrian, and 120 Swiss centers. RESULTS: The survey was completed by 183 of the 1412 centers surveyed (12.7% response rate). Most centers (90.2%) do not conduct a test shock to confirm the integrity of the HV circuit before decision-making. Procedural practice in lead management varies depending on the presentation of lead failure and whether the center applies a test shock. In centers that do not conduct a test shock, the majority (69.9%) implant a new HV-P/S lead. Most centers (61.7%) that test the integrity of the HV system implant a P/S lead. The majority of centers favor DF-4 connectors (74.1%) over DF-1 connectors (25.9%) at first CIED implantation. CONCLUSION: Either implanting a new HV-P/S lead or placing an additional P/S lead are selected strategies if the implantable cardioverter-defibrillator lead failure is localized to the P/S portion. However, conducting a test shock to confirm the integrity of the HV component is rarely performed.

Evaluation of insertion-deletion markers suitable for genetic diversity studies and marker-trait correlation analyses in cultivated peanut (Arachis hypogaea L.).

Peanut is one of the most important oil crops worldwide. We used insertion-deletion (InDel) markers to assess the genetic diversity and population structure in cultivated peanut. Fifty-four accessions from North China were genotyped using 48 InDel markers. The markers amplified 61 polymorphic loci with 1 to 8 alleles and an average of 2.6 alleles per marker. The polymorphism information content values ranged from 0.0364 to 0.9030, with an average of 0.5038. Population structure and neighbor-joining (NJ) tree analyses suggested that all accessions could be divided into four clusters (A1-A4), using the NJ method. Likewise, four subpopulations (G1-G4) were identified using STRUCTURE analysis. A principal component analysis was also used and results concordant with the other analysis methods were found. A multi-linear stepwise regression analysis revealed that 13 InDel markers correlated with five measured agronomical traits. Our results will provide important information for future peanut molecular breeding and genetic research.

Methanosarcinaceae and acetate-oxidizing pathways dominate in high-rate thermophilic anaerobic digestion of waste-activated sludge.

This study investigated the process of high-rate, high-temperature methanogenesis to enable very-high-volume loading during anaerobic digestion of waste-activated sludge. Reducing the hydraulic retention time (HRT) from 15 to 20 days in mesophilic digestion down to 3 days was achievable at a thermophilic temperature (55°C) with stable digester performance and methanogenic activity. A volatile solids (VS) destruction efficiency of 33 to 35% was achieved on waste-activated sludge, comparable to that obtained via mesophilic processes with low organic acid levels (<200 mg/liter chemical oxygen demand [COD]). Methane yield (VS basis) was 150 to 180 liters of CH4/kg of VS(added). According to 16S rRNA pyrotag sequencing and fluorescence in situ hybridization (FISH), the methanogenic community was dominated by members of the Methanosarcinaceae, which have a high level of metabolic capability, including acetoclastic and hydrogenotrophic methanogenesis. Loss of function at an HRT of 2 days was accompanied by a loss of the methanogens, according to pyrotag sequencing. The two acetate conversion pathways, namely, acetoclastic methanogenesis and syntrophic acetate oxidation, were quantified by stable carbon isotope ratio mass spectrometry. The results showed that the majority of methane was generated by nonacetoclastic pathways, both in the reactors and in off-line batch tests, confirming that syntrophic acetate oxidation is a key pathway at elevated temperatures. The proportion of methane due to acetate cleavage increased later in the batch, and it is likely that stable oxidation in the continuous reactor was maintained by application of the consistently low retention time.

Determination and kinetics of enrofloxacin and ciprofloxacin in Tra catfish (Pangasianodon hypophthalmus) and giant freshwater prawn (Macrobrachium rosenbergii) using a liquid chromatography/mass spectrometry method.

Determination and kinetics of enrofloxacin and ciprofloxacin in Tra catfish (Pangasianodon hypophthalmus) and giant freshwater prawn (Macrobrachium rosenbergii) using a liquid chromatography/mass spectrometry method. J. vet. Pharmacol. Therap. 34, 142-152. The fluoroquinolones enrofloxacin (EF) and ciprofloxacin (CF) residues were investigated in the edible tissues of two important Asian aquacultured species such as Tra catfish (Pangasianodon hypophthalmus) and giant freshwater prawn (Macrobrachium rosenbergii) using a sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry method. Fish and prawn were treated with medicated feed with multiple doses of EF, in field conditions. A validation study of the analytical method was realized in terms of linearity, specificity, precision (repeatability and within-laboratory reproducibility), recovery and decision limit (CCα). The time needed before the antibiotic disappears from animal tissues or reach the maximum residue limit (MRL, 100µg/kg) was assessed. The concentration values of EF detected in Tra catfish tissue were between the MRL and 2×MRL concentrations, according to the fish density, 7days following the end of the enrofloxacin treatment (20mg/kg body weight per day, for seven consecutive days). The concentration value of ER in prawn tissue was lower than the MRL and the limit of quantification (LOQ, 14µg/kg) 5 and 7days after the stop of the EF treatment (50mg/kg body weight per day, for five consecutive days), respectively. The mean detected levels of CF was much lower in comparison with that of EF, indicating that only a small part of EF is metabolized into CF (<5%) in both Tra catfish and prawn.

SCAMP3 is a mutant EGFR phosphorylation target and a tumor suppressor in lung adenocarcinoma.

Mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain constitutively activate EGFR resulting in lung tumorigenesis. Activated EGFR modulates downstream signaling by altering phosphorylation-driven interactions that promote growth and survival. Secretory carrier membrane proteins (SCAMPs) are a family of transmembrane proteins that regulate recycling of receptor proteins, including EGFR. The potential role of SCAMPs in mutant EGFR function and tumorigenesis has not been elucidated. Using quantitative mass-spectrometry-based phosphoproteomics, we identified SCAMP3 as a target of mutant EGFRs in lung adenocarcinoma and sought to further investigate the role of SCAMP3 in the regulation of lung tumorigenesis. Here we show that activated EGFR, either directly or indirectly phosphorylates SCAMP3 at Y86 and this phosphorylation increases the interaction of SCAMP3 with both wild-type and mutant EGFRs. SCAMP3 knockdown increases lung adenocarcinoma cell survival and increases xenograft tumor growth in vivo, demonstrating a tumor suppressor role of SCAMP3 in lung tumorigenesis. The tumor suppressor function is a result of SCAMP3 promoting EGFR degradation and attenuating MAP kinase signaling pathways. SCAMP3 knockdown also increases multinucleated cells in culture, suggesting that SCAMP3 is required for efficient cytokinesis. The enhanced growth, increased colony formation, reduced EGFR degradation and multinucleation phenotype of SCAMP3-depleted cells were reversed by re-expression of wild-type SCAMP3, but not SCAMP3 Y86F, suggesting that Y86 phosphorylation is critical for SCAMP3 function. Taken together, the results of this study demonstrate that SCAMP3 functions as a novel tumor suppressor in lung cancer by modulating EGFR signaling and cytokinesis that is partly Y86 phosphorylation-dependent.

Alterations in the Global Proteome and Phosphoproteome in Third Generation EGFR TKI Resistance Reveal Drug Targets to Circumvent Resistance.

Lung cancer is the leading cause of cancer mortality worldwide. The treatment of patients with lung cancer harboring mutant EGFR with orally administered EGFR tyrosine kinase inhibitors (TKI) has been a paradigm shift. Osimertinib and rociletinib are third-generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard of care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here we characterized the proteome and phosphoproteome of a series of isogenic EGFR-mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs, comprising the most comprehensive proteomic dataset resource to date to investigate third generation EGFR TKI resistance in lung adenocarcinoma. Unbiased global quantitative mass spectrometry uncovered alterations in signaling pathways, revealed a proteomic signature of epithelial-mesenchymal transition, and identified kinases and phosphatases with altered expression and phosphorylation in TKI-resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition, resulting in subsequent inhibition of RAS/MAPK and activation of PI3K/AKT pathways. Anticorrelation analyses of this phosphoproteomic dataset with published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures program predicted drugs with the potential to overcome EGFR TKI resistance. The PI3K/MTOR inhibitor dactolisib in combination with osimertinib overcame resistance both in vitro and in vivo. Taken together, this study reveals global proteomic alterations upon third generation EGFR TKI resistance and highlights potential novel approaches to overcome resistance. SIGNIFICANCE: Global quantitative proteomics reveals changes in the proteome and phosphoproteome in lung cancer cells resistant to third generation EGFR TKIs, identifying the PI3K/mTOR inhibitor dactolisib as a potential approach to overcome resistance.

Data mining cDNAs reveals three new single stranded RNA viruses in Nasonia (Hymenoptera: Pteromalidae).

We report three novel small RNA viruses uncovered from cDNA libraries from parasitoid wasps in the genus Nasonia. The genome of this kind of virus is a positive-sense single-stranded RNA with a 3' poly(A), which facilitates cloning from cDNAs. Two of the viruses, NvitV-1 and NvitV-2, possess a RNA-dependent RNA polymerase that associates them with the family Iflaviridae of the order Picornavirales. A third virus, NvitV-3, is most similar to the Nora virus from Drosophila. A reverse transcription-PCR method developed for NvitV-1 indicates that it is a persistent commensal infection of Nasonia.

Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair.

Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation, directed by local morphogen signals and mechanical cues. Here, we aimed to mimic development for regeneration of large bone defects. We hypothesized that engineered human mesenchymal condensations presenting transforming growth factor-ß1 (TGF-ß1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles promotes endochondral defect regeneration contingent on in vivo mechanical cues. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with BMP-2 + TGF-ß1 fully restoring mechanical function. Delayed in vivo ambulatory loading significantly enhanced the bone formation rate in the dual morphogen group. In vitro, BMP-2 or BMP-2 + TGF-ß1 initiated robust endochondral lineage commitment. In vivo, however, extensive cartilage formation was evident predominantly in the BMP-2 + TGF-ß1 group, enhanced by mechanical loading. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.

Species-diagnostic single-nucleotide polymorphism and sequence-tagged site markers for the parasitic wasp genus Nasonia (Hymenoptera: Pteromalidae).

Wasps of the genus Nasonia are important biological control agents of house flies and related filth flies, which are major vectors of human pathogens. Species of Nasonia (Hymenoptera: Pteromalidae) are not easily differentiated from one another by morphological characters, and molecular markers for their reliable identification have been missing so far. Here, we report eight single-nucleotide polymorphism and three sequence-tagged site markers derived from expressed sequenced tag libraries for the two closely related and regionally sympatric species N. giraulti and N. vitripennis. We studied variation of these markers in natural populations of the two species, and we mapped them in the Nasonia genome. The markers are species-diagnostic and evenly spread over all five chromosomes. They are ideal for rapid species identification and hybrid recognition, and they can be used to map economically relevant quantitative trait loci in the Nasonia genome.

The clinical significance of ABCB1 overexpression in predicting outcome of CML patients undergoing first-line imatinib treatment.

Tyrosine kinase inhibitor (TKI) therapy results in excellent responses in the majority of patients with chronic myeloid leukaemia. First-line imatinib treatment, with selective switching to nilotinib when patients fail to meet specific molecular targets or for imatinib intolerance, results in excellent overall molecular responses and survival. However, this strategy is less effective in cases of primary imatinib resistance; moreover, 25% of patients develop secondary resistance such that 20-35% of patients initially treated with imatinib will eventually experience treatment failure. Early identification of these patients is of high clinical relevance. Since the drug efflux transporter ABCB1 has previously been implicated in TKI resistance, we determined if early increases in ABCB1 mRNA expression (fold change from diagnosis to day 22 of imatinib therapy) predict for patient response. Indeed, patients exhibiting a high fold rise (⩾2.2, n=79) were significantly less likely to achieve early molecular response (BCR-ABL1IS ⩽10% at 3 months; P=0.001), major molecular response (P<0.0001) and MR4.5 (P<0.0001). Additionally, patients demonstrated increased levels of ABCB1 mRNA before the development of mutations and/or progression to blast crisis. Patients with high fold rise in ABCB1 mRNA were also less likely to achieve major molecular response when switched to nilotinib therapy (49% vs 82% of patients with low fold rise). We conclude that early evaluation of the fold change in ABCB1 mRNA expression may identify patients likely to be resistant to first- and second-generation TKIs and who may be candidates for alternative therapy.

Vasoactive intestinal peptide dampens formyl-peptide-induced ROS production and inflammation by targeting a MAPK-p47phox phosphorylation pathway in monocytes.

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47phox, p67phox, p40phox, and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA11,22,28]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the "MAPK-p47phox phosphorylation-NOX2 activation" axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.

Effective elimination of liver cancer stem-like cells by CD90 antibody targeted thermosensitive magnetoliposomes.

AIM: To investigate the use of thermosensitive magnetoliposomes (TMs) loaded with magnetic iron oxide (Fe3O4) and the anti-cancer stem cell marker CD90 (CD90@TMs) to target and kill CD90+ liver cancer stem cells (LCSCs). METHODS: The hepatocellular carcinoma cell line Huh7 was used to separate CD90+ LCSCs by magnetic-activated cell sorting. CD90@TMs was characterized and their ability to target CD90+ LCSCs was determined. Experiments were used to investigate whether CD90@TMs combined with magnetic hyperthermia could effectively eliminate CD90+ LCSCs. RESULTS: The present study demonstrated that CD90+ LCSCs with stem cells properties were successfully isolated. We also successfully prepared CD90@TMs that was almost spherical and uniform with an average diameter of 130±4.6 nm and determined that magnetic iron oxide could be incorporated and retained a superparamagnetic response. CD90@TMs showed good targeting and increased inhibition of CD90+ LCSCs in vitro and in vivo compared to TMs. CONCLUSIONS: CD90@TMs can be used for controlled and targeted delivery of anticancer drugs, which may offer a promising alternative for HCC therapy.

VP-16 and cisplatin as first-line therapy for small-cell lung cancer.

Thirty-one patients with small-cell lung cancer (SCLC) were treated with VP-16 and cisplatin as first-line therapy. In the majority of cases an Adriamycin (Adria Laboratories, Columbus, Ohio) containing regimen was contraindicated because of severe cardiac or hepatic disease. Eight patients who presented with cerebral metastases were also included in the series. Eleven patients had limited disease (LD), and 20 had extensive disease (ED). Of the 28 evaluable patients, 12 (43%) achieved a complete response (CR) and 12 (43%) had a partial response (PR). Four patients (14%) either had no response or progressed on treatment. The median duration of response for patients with LD was 39 weeks and for those with ED, 26 weeks. The median survival time (MST) for the whole group of responding (CR and PR) LD patients was 70 weeks (range, 28 to 181 + weeks), and for responding ED patients, it was 43 weeks (range, 17 to 68 weeks). Gastrointestinal toxicity was mild, but leukopenia and thrombocytopenia were common. There were four febrile episodes during periods of drug-induced neutropenia and this led to one treatment-related death. Nephrotoxicity occurred in 15 patients and required discontinuation of cisplatin in two. These results compare favorably with reports of standard induction chemotherapy regimens and provide further evidence of the activity of the VP-16 and cisplatin regimen in patients with SCLC.

Chemotherapy following surgery for stages IE and IIE non-Hodgkin's lymphoma of the gastrointestinal tract.

Twenty-six patients were treated with chemotherapy following surgery for gastrointestinal non-Hodgkin's lymphoma (GI-NHL). The median age was 50 years (range, 20 to 76). The primary site included stomach (16 patients), small bowel (seven), large bowel (two), and mesenteric nodes (one). Following surgery, nine patients had macroscopic and four patients had microscopic residual disease, and 13 were felt to have had complete surgical resection. Thirteen patients were stage I and 13 were stage II. Sixteen patients were treated with COPP (cyclophosphamide, vincristine, procarbazine, prednisone), nine with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), and one with CVP (cyclophosphamide, vincristine, prednisone). At a median follow-up of 50 months (8+ to 178+ months) ten of 12 stage I patients and nine of 14 stage II patients remain alive. Of the nine patients with macroscopic residual disease, four died of disease 6.5 to 11.0 months after diagnosis, and five are alive 8+ to 178+ months from diagnosis. Fourteen of the remaining 17 patients who had complete surgical resection are alive without disease. Death in the other three patients was due to multiple abdominal abscesses at 12 months, adenocarcinoma of the colon at 57 months, and dementia and progressive neurologic dysfunction at 65 months. No patient who had complete resection has relapsed or developed systemic disease after chemotherapy. These results suggest that complete surgical resection is an important prognostic factor and that chemotherapy without irradiation in completely resected localized GI-NHL can prevent local and systemic relapse resulting in long-term disease-free survival.

Forskolin mimics TSH action on the expression of protein kinase C isozymes in pig thyroid cell cultures.

In porcine thyroid cell cultures, phospholipid-dependent protein kinases (PKCs) have the same characteristics as intact glands. The overall PKC activity, presence of PKC isozymes, chromatographic pattern and endogenous substrates specificity were not modified during the two-day culture period. Three PKC isozymes (cPKC epsilon, nPKC epsilon and aPKC zeta) were identified by immunoblot analysis in the two subcellular fractions: cytosol and particulate extract, both in intact glands and two-day-old cultures. In cells cultured for two days in the presence of TSH (0.1 mU/ml), the overall PKC activity was stimulated (ca. 200%) in the two compartments. This stimulation was parallel to the increase in protein expression of the three PKC isoforms (as demonstrated by immunoblot analysis) and was accompanied by a redistribution of cPKC alpha and nPKC epsilon toward the particulate fraction. In TSH-treated cells, hydroxyapatite chromatography of cytosolic PKC revealed an additional peak of PKC activity eluted at 195 mM potassium phosphate. Its elution molarity did not correspond to the molarity of any known PKC isozyme, and it did not cross-react with antibodies directed against cPKC isozymes--: alpha, beta, or gamma. When TSH was replaced by forskolin (10(-5) M), identical quantitative and qualitative modifications were obtained, suggesting that, in thyroid cells, the cyclic AMP-dependent regulatory cascade could be involved in the control of PKC isoforms expression by TSH.

P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox.

NADPH oxidase is an O2*- -generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67phox, p47phox, p40phox, and the G-protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67phox, p47phox, and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40phox associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40phox. When resting neutrophils were lysed in Triton X-100 or octyl glucoside buffer and separated into detergent-soluble and detergent-insoluble fractions, p40phox and p67phox were mainly associated with the detergent-insoluble fraction (defined as the cytoskeleton), whereas p47phox was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47phox translocation to the cytoskeleton but did not affect the distribution of p40phox or p67phox. Using immunofluorescence confocal microscopy, we found that p40phox colocalized with filamentous actin. In neutrophils from a p67phox-deficient patient with detectable p40phox, p40phox associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X-100 buffer, p40phox, p47phox, and p67phox were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40phox and p47phox was found in the soluble membrane fraction, but no p67phox was detected. These findings show that p40phox, like p67phox and p47phox, binds to the cytoskeleton and membrane skeleton. In addition, p40phox can dissociate from p67phox in activated membranes.

Identification of Transcripts Involved in Resistance Responses to Leaf Spot Disease Caused by Cercosporidium personatum in Peanut (Arachis hypogaea).

ABSTRACT Late leaf spot disease caused by Cercosporidium personatum is one of the most destructive foliar diseases of peanut (Arachis hypogaea) worldwide. The objective of this research was to identify resistance genes in response to leaf spot disease using microarray and real-time polymerase chain reaction (PCR). To identify transcripts involved in disease resistance, we studied the gene expression profiles in two peanut genotypes, resistant or susceptible to leaf spot disease, using cDNA microarray containing 384 unigenes selected from two expressed sequenced tag (EST) cDNA libraries challenged by abiotic and biotic stresses. A total of 112 spots representing 56 genes in several functional categories were detected as up-regulated genes (log(2) ratio > 1). Seventeen of the top 20 genes, each matching gene with known function in GenBank, were selected for validation of their expression levels using real-time PCR. The two peanut genotypes were also used to study the functional analysis of these genes and the possible link of these genes to the disease resistance trait. Microarray technology and real-time PCR were used for comparison of gene expression. The selected genes identified by microarray analysis were validated by real-time PCR. These genes were more greatly expressed in the resistant genotype as a result of response to the challenge of C. personatum than in the susceptible genotype. Further investigations are needed to characterize each of these genes in disease resistance. Gene probes could then be developed for application in breeding programs for marker-assisted selection.

Immunochemical identification and translocation of protein kinase C zeta in human neutrophils.

Western blots of human polymorphonuclear leukocyte (PMN) extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. Two bands corresponding to PKC type zeta with apparent molecular masses of 81 kDa and 76 kDa were identified in the cytosolic fraction of resting cells, in addition to PKC types alpha and beta. PKC zeta was apparently abundant, like PKC beta, whereas PKC delta, -epsilon, and -gamma were not detectable. Following short stimulation (5 min) of PMN with phorbol-12-myristate-13-acetate (1 microgram/ml), physical translocation of PKC zeta from the cytosol to the plasma membrane fraction occurred, although this isoform does not bind phorbol esters. These data show that, in addition to the two calcium-dependent isoenzymes alpha and beta, human PMN express a calcium-independent isoenzyme zeta which translocates in stimulated cells, suggesting a role in the regulation of antibacterial activities.

Decreased duodenal enterocyte calcium flux rates in the spontaneously hypertensive rat.

We have previously reported serum 1,25(OH)2 vitamin D3 (calcitriol) and active transduodenal calcium absorption measured in the Ussing chamber to be reduced in 12- to 14-week-old male Okamoto-Aoki spontaneously hypertensive rats (SHR). In the present study, we compared rates of calcium influx and efflux in isolated duodenal enterocytes in SHR and corresponding controls, Wistar-Kyoto rats (WKY). The early (0 to 1 minute) and the late (1 to 15 minute) phase of calcium influx rates at 1.0 mmol Ca2+ in the incubation medium were lower in the SHR than in the WKY (mean +/- SEM): 1.93 +/- 0.22 v 2.85 +/- 0.41 nmol/mg protein/min, n = 8 and n = 7 experiments, respectively, P less than .05; and 0.334 +/- 0.025 v 0.488 +/- 0.059 nmol/mg protein/min, n = 14 pairs, P less than .01. The calcium efflux rate constant of the SHR was reduced: 34.3 +/- 1.4 v 51.9 +/- 1.4% per hour, n = 11 pairs, P less than .01. However, in the absence of sodium or the presence of ouabain (4.0 mmol) in the incubation medium, a decrease in this constant was observed in the WKY but not in the SHR. These data, at the cellular level, support our previous observation in intact tissue of reduced active transduodenal calcium transport in the 12- to 14-week-old SHR. Whether the primary defect in calcium transport involves the luminal or the basolateral membrane of the enterocyte, or whether both disturbances are due to a common primary perturbation cannot be deduced from the present experiments.