Additional Spot Research Articles

PQQ‐Dependent Alcohol Dehydrogenase (QEDH) of Pseudomonas aeruginosa is involved in catabolism of acyclic terpenes

Growth of Pseudomonas aeruginosa on acyclic terpene alcohols such as geraniol depends on the presence of the atuRABCDEFGH gene cluster and a functional acyclic terpene utilisation (Atu) pathway. The proteins encoded by the atu gene cluster are necessary but not sufficient for growth on acyclic terpenes. Comparative 2-dimensional polyacrylamide gel electrophoresis of soluble P. aeruginosa proteins revealed the presence of an additional spot (besides Atu proteins) that is specifically expressed in geraniol cells but is absent in isovalerate-grown cells. The spot was identified as PA1982 gene product a pyrroloquinoline quinone (PQQ) dependent ethanol oxidoreductase (QEDH). Inactivation of PA1982 by insertion mutagenesis resulted in inability of the mutant to utilise ethanol and in reduced growth on geraniol. Growth on ethanol was restored by transferring an intact copy of the PA1982 gene into the mutant. The PA1982 gene product was purified from recombinant Escherichia coli and revealed PQQ-dependent oxidoreductase activity with a variety of substrates including acyclic terpene derivates at comparable V(max)-values. Our results show that QEDH participates in oxidation of acyclic terpene derivates in addition to the well-known function in ethanol metabolism.

EDTA in Dried Blood Spots Leads to False Results in Neonatal Endocrinologic Screening

Blood samples for neonatal screening for inborn errors of metabolism are collected and shipped on standardized filter paper cards. Occasionally these samples are contaminated with EDTA, which is often used for anticoagulation. EDTA may interfere with newborn screening tests based on lanthanide fluorescence and thus lead to false-negative or false-positive results. We used tandem mass spectrometry (MS/MS) to detect EDTA in dried blood spots by use of an extra experiment that was integrated into the standard MS/MS neonatal screening and did not require an additional sample spot, nor extra time or work. We analyzed the influence of different blood sampling procedures on lanthanide fluorescence tests for thyroid-stimulating hormone (TSH) and 17-hydroxyprogesterone (17-OHP). EDTA was increased in 138 of 190 000 newborn screening samples, 27 of which caused false- positive results in the immunoassay for 17-OHP. No false-negative TSH results were found. False-positive results in the 17-OHP test occurred when EDTA concentrations were >2.0 g/L; the TSH test, however, produced false negatives only when EDTA concentrations were >3.0 g/L. Using EDTA-containing devices the procedure of blood collection significantly influenced the concentration of the anticoagulant. Addition of EDTA quantification into standard MS/MS tests is a simple and useful method to avoid false-positive or false-negative neonatal screening results in lanthanide fluorescence-based tests.

Novel druggable hot spots in avian influenza neuraminidase H5N1 revealed by computational solvent mapping of a reduced and representative receptor ensemble.

The influenza virus subtype H5N1 has raised concerns of a possible human pandemic threat because of its high virulence and mutation rate. Although several approved anti-influenza drugs effectively target the neuraminidase, some strains have already acquired resistance to the currently available anti-influenza drugs. In this study, we present the synergistic application of extended explicit solvent molecular dynamics (MD) and computational solvent mapping (CS-Map) to identify putative ‘hot spots’ within flexible binding regions of N1 neuraminidase. Using representative conformations of the N1 binding region extracted from a clustering analysis of four concatenated 40-ns MD simulations, CS-Map was utilized to assess the ability of small, solvent-sized molecules to bind within close proximity to the sialic acid binding region. Mapping analyses of the dominant MD conformations reveal the presence of additional hot spot regions in the 150- and 430-loop regions. Our hot spot analysis provides further support for the feasibility of developing high-affinity inhibitors capable of binding these regions, which appear to be unique to the N1 strain.

Combining superior agronomic performance and terminal heat tolerance with resistance to spot blotch ( Bipolaris sorokiniana) of wheat in the warm humid Gangetic Plains of South Asia

Seven hundred twenty-nine lines of diverse wheat germplasm lines were evaluated in eight locations of three countries (India, Nepal and Bangladesh) of South Asia for 5 years (1999–2000 to 2003–2004) through Eastern Gangetic Plains Screening Nursery (EGPSN) organized by CIMMYT South Asia, Nepal, for agronomic performance and tolerance to spot blotch of wheat. Each year, the number of lines represented a new set of 150 lines that included six common checks and a different local check at each of the eight locations. One hundred and five lines, 21 in each year, advanced from EGPSN were also tested for 5 years (2000–2001 to 2004–2005) in five locations of South Asia through Eastern Gangetic Plains Yield Trials (EGPYT) to verify spot blotch tolerance and superior yield performance of the selected germplasm. Many lines yielded significantly more than the best check and possessed high levels of spot blotch resistance under warm humid environments of South Asia. The most promising 25 lines have been listed as sources of strong resistance, with 9 lines better yielding than the best resistant check PBW 343 in fewer days to maturity. Most of these superior lines represented elite CIMMYT germplasm and around half were derived from Kauz and Veery. The line EGPYT 67, Kauz//Kauz/Star/3/Prinia/4/Milan/Kauz, was the best for spot blotch resistance, yield, days to maturity, and 1000 grain weight (TKW). The next two lines in the order of merit were EGPYT 84 (Mrng/Buc//Blo/Pvn/3/Pjb 81) and EGPYT 69 (Chirya3/Pastor). The results demonstrate that additional spot blotch resistant wheat genotypes with high grain yield and TKW, and early maturity, have become available as a result of the regional and international collaboration in South Asia.

Polyunsaturated fatty acid suppression of fatty acid synthase (FASN): evidence for dietary modulation of NF-Y binding to the Fasn promoter by SREBP-1c

Dietary PUFAs (polyunsaturated fatty acids) co-ordinately suppress transcription of a group of hepatic genes encoding glycolytic and lipogenic enzymes. Suppression of Fasn (fatty acid synthase) transcription involves two PUFA-responsive regions, but the majority of PUFA sensitivity maps to a region within the proximal promoter containing binding sites for NF-Y (nuclear factor-Y), Sp1 (stimulatory protein 1), SREBP (sterol-regulatory-elementbinding protein), and USF (upstream stimulatory factor). Promoter activation assays indicate that altered NF-Y is the key component in regulation of Fasn promoter activity by PUFA. Using electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis, we demonstrate for the first time that PUFAs decrease in vivo binding of NF-Y and SREBP-1c to the proximal promoter of the hepatic Fasn gene and the promoters of three additional genes, spot 14, stearoyl-CoA desaturase and farnesyl diphosphate synthase that are also down-regulated by PUFA. The comparable 50% decrease in NF-Y and SREBP-1c binding to the promoters of the respective PUFA-sensitive genes occurred despite no change in nuclear NF-Y content and a 4-fold decrease in SREBP-1c. Together, these findings support a mechanism whereby PUFA reciprocally regulates the binding of NF-Y and SREBP-1c to a subset of genes which share similar contiguous arrangements of sterol regulatory elements and NF-Y response elements within their promoters. PUFA-dependent regulation of SREBP-1c and NF-Y binding to this unique configuration of response elements may represent a nutrient-sensitive motif through which PUFA selectively and co-ordinately targets subsets of hepatic genes involved in lipid metabolism.

A report on the incidence of intestinal 99mTc-methylene diphosphonate uptake of bone scans and a review of the literature

In addition to well-known specific conditions for soft-tissue uptake of bone-seeking radiotracers, there is a limited number of reports on intestinal uptake of (99m)Tc-methylene diphosphonate ((99m)Tc-MDP) on bone scans. To describe the incidence of intestinal accumulation of (99m)Tc-MDP on bone scans in adult patients, define the patterns of this unusual finding and review the literature on its causes. Two thousand, one hundred and forty-four consecutive patients have been evaluated for intestinal (99m)Tc-MDP uptake on bone scans. Intestinal uptake was observed visually 3-4 h after the administration of the radiopharmaceutical. A whole-body bone scan and various spot views of the abdomino-pelvic region were obtained with a dual-headed gamma camera to evaluate the intestinal uptake. Delayed scans were also obtained as well as co-relative imaging and/or colonoscopic studies in some of intestinal uptake patients. Six patients had delayed scans of the abdomino-pelvic region. Fourteen patients had comparable scans either a year before or a year later. The positive intestinal uptake scans were further grouped according to the localization and intensity (mild uptake: lower than iliac bone; moderate uptake: equal to iliac bone; significant uptake: higher than iliac bone). Twenty-two (17 female, five male) patients out of 2144 with a mean age of 57 years showed intestinal (99m)Tc-MDP uptake. The localization was mainly (20/22) in the right abdomino-pelvic region projecting on and in the configuration of ascending colon while one patient showed intestinal uptake all over the abdomen and one displayed diffuse intestinal radioactivity in his right hemithorax. The majority of the cases showed moderate to intense intestinal uptake (18/22). Six patients showed a decrease, disappearance or alteration in the intestinal uptake on the delayed images. Re-evaluation bone scans in five patients 1 year later showed no intestinal uptake this time. Among nine patients with prior bone scans 1 year before, intestinal uptake was negative in seven at that time. No significant pathology was obtained on the correlative images. (99m)Tc-MDP uptake can be observed in the intestines in 1% of bone scans with a prominent localization in the ascending colon and rarely all over the intestines or in thorax due to Chilaiditi's syndrome, as well. The mechanism of intestinal uptake is still unclear in some of the patients. Delayed imaging, additional spot views and SPECT studies help in the differentiation of this finding from possible misinterpretation. Intestinal (99m)Tc-MDP uptake on bone scan could be an intermittent process and should be included among other well-known reasons of soft-tissue uptake.

Temporal Variability in Diet and Urinary Concentrations of Pyrethroid and Organophosphate Pesticide Metabolites in Adult Volunteers From a Large U.S. City

P-354 Introduction: We are investigating the validity of using urinary biomarkers to estimate dietary exposures to pyrethroid and organophosphate pesticides in a pilot sample of adult volunteers (n = 11) from a large urban area of the United States. This information is needed to help determine the most accurate way of measuring exposures to non-persistent pesticides among the general adult population. Methods: We collected four consecutive days of duplicate diet samples and 10 spot urine samples from each subject. First morning void and before bedtime spot urine samples were collected on each day of diet sampling. Additional spot urines were collected the night before the first day of diet sampling and the morning after the last day of diet sampling. Detailed diet information including food description, organic status, preparation details (e.g. raw vs. cooked, peeled vs. unpeeled, etc.) and weight (in grams) was collected for each item consumed over the four-day sampling period. The four-day protocol was repeated twice for each subject, once in summer (July–October 2005) and once in winter (January–March 2006), to examine seasonal differences. Urine samples are currently being analyzed for selected pyrethroid and organophosphate metabolites while method development is underway for quantifying parent pesticide residues in the duplicate diet samples. Results: We present findings on intra- and inter-subject variability in urinary metabolite concentrations across the four-day sampling period, and across the summer and winter sampling cycles. We also present results of multivariate longitudinal regression analyses to determine which food items are most significantly associated with urinary metabolite concentrations among subjects in our pilot sample. Discussion/conclusions: This is the first study we know of to date to measure food intake and urinary pyrethroid and organophosphate metabolites in a multi-day longitudinal sample of non-occupationally exposed adults. For food intakes showing no association, alternative strategies for measuring dietary exposure to non-persistent pesticides must be considered. For those displaying significant association, urinary levels offer a valid tool for estimating dietary exposures among similar adult populations. This study will also provide important reference data on short-term temporal variability in urinary pyrethroid and organophosphate metabolite concentrations among U.S. adults.

Urban Drainage Network Generation with Geographic Information Systems Using Remotely Sensed Data

In Sri Lanka most of the existing urban drainage systems are not functioning properly. Establishing a proper drainage system in an urban area.is extremely important since it could avoid floods and inundation which causes damage to property and inconvenience to city dwellers; improve health conditions of the urban community by avoiding stagnant water; give an aesthetically pleasing environment, etc. Therefore, in Sri Lanka, establishing a proper drainage system and maintaining the same has become a major challenge in urban areas. Urban drainage network identification is a primary requirement for developing a proper drainage system. Urban drainage network identification is a tedious task, which is usually done through engineering surveys. The computer aided Geographical Information Systems (GIS) can be utilized to generate drainage network from the terrain data. The main objective of this work is the study of stream network generation in GIS using data extracted from aerial photographs, carryout accuracy comparison with surveyed drainage network and then to identify the parameters that affect the accuracy of generating streamlines. In this study, two urban watersheds from Colombo and one watershed from Moratuwa were studied to identify the stream network generation and associated terrain indicators using data extracted from aerial photographs. The data extraction for the study was done using three methods. Existing drainage network and watershed boundary were digitized from hard copy maps of Sri Lanka Land Reclamation and Development Corporation (SLLRDC). Contours, spot heights, buildings and road data in digital form were extracted from the digital archives of the National Survey Department (NSD). Some of the existing streamlines and culvert locations were surveyed specifically using GPS. Then the generation of the Triangulated Irregular Network (TIN) was carried out using the extracted contours and spot heights. Using the TIN, Digital Elevation Models (DEM) for spatial resolutions of 2m, 5m, 10m, 20m & 50m were generated. For each DEM, flow direction grids and flow accumulation grids were generated as components of stream network generation. Once the streamlines are generated from the flow accumulation grid, it is necessary to give a threshold value to separate the stream network. After comparing several vector and grid based methods, it was identified that Grid Based Comparative Squares (GBCS) method could be effectively used for comparing the accuracy of generated and extracted streamlines. In the GBCS method the streamlines generated and observed are matched for fitting only within identified square areas. In the squares, comparison is done using zones of distance from observed streamlines created using buffering capability of GIS, and giving an error code for the generated lines which fall into buffers that represent the deviation. Therefore, once the squares are selected spatially, then the degree of fitting of the computed and the observed streamline are compared with a system of buffers drawn to each observed streamline within a square. The squares and a buffer zoning of the extracted streams enable the identification of deviations of the generated streams from those of engineering survey sheets. Deviations were compared using RMSE (Root Mean Square Error) as the numerical indicator. The extracted stream network of catchments indicated a strong human influence in deviating the stream network and hence buildings were combined to the DEM to identify whether the results would show a difference. The same accuracy assessment method for five spatial resolutions was used for comparison of stream network. Also Flatness, Stream Order and Surface Slope based on streamline network of each watershed were computed for comparison. The study also identified that the accuracy levels were lower in the areas of lower stream order. This shows that in the upper reaches the streamlines would vary significantly probably due to less contributing cells and also as a result of human interventions. The stream network identification to a reasonable accuracy using remotely sensed data of 0.4m accurate contours and additional spot heights is not a straightforward operation in the case of selected watersheds. The significant flat terrain poses problems in identifying the flow directions to find a unique stream network.

Vibrio choleraeSXT Element, Laos

<i>Vibrio cholerae</i>SXT Element, Laos

Survival Analysis of Time to Abscission of Blueberry Leaves Affected by Septoria Leaf Spot

ABSTRACT In the southeastern United States, Septoria leaf spot, caused by Septoria albopunctata, can result in premature defoliation of blueberry plants during summer and fall, thereby reducing yield potential for the following year. The effects of disease severity and leaf attributes (leaf age and leaf location in the canopy) on the dynamics (timing and extent) of defoliation were quantified in field plots of Premier rabbiteye blueberry (Vaccinium ashei) in 2002 and 2003. In each year, 50 shoots were selected for assessment in early spring, and all leaves on these shoots (n = 410 and 542 in 2002 and 2003, respectively) were monitored individually for disease progress and time of abscission at 3- to 10-day intervals throughout the season. In both years, disease progress was characterized by an exponential increase in disease severity up to late September, followed by a decline toward the end of the assessment period in late November. Defoliation was sporadic up to late August, followed by more rapid and sustained levels of leaf loss. Abscission of severely infected leaves could explain the decline in disease severity toward the end of the season. Final disease severity (i.e., disease severity on the last assessment date before leaf drop) was highest for leaves that abscised early and lowest for leaves that had not abscised by the end of the assessment period. Survival analysis revealed that older leaves (located on the lower halves of shoots) and leaves with high levels of disease (>/=5 spots/leaf at the time of fruit harvest in mid-June) abscised significantly (P < 0.0001) earlier than younger leaves and leaves with lower disease severity. Relative to their respective reference groups, mean times to abscission were approximately 2 weeks shorter for the older leaf group and approximately 3 weeks shorter in the leaf group afflicted by high disease severity. When an accelerated failure time model was fitted to the data, the resulting parameter estimates indicated that each additional leaf spot present at harvest accelerated time to leaf abscission (expressed using late August as a starting point) by 1.9 and 4.5% in 2002 and 2003, respectively. Leaf location in upper or lower portions of the canopy had no significant effect on time to abscission (P > 0.05).

Further Studies on the Hyperphosphorylated Form (p40) of the Rabies Virus Nominal Phosphoprotein (P)

We investigated possible mechanisms involved in production of a hyperphosphorylated form (p40) of rabies virus P protein, to which two dimensional (2-D) gel electrophoresis was applied. The P gene products produced in Escherichia coli cells could be detected as a single spot of unphosphorylated 37-kDa form (termed as p37-0) in a 2-D gel. The 37-kDa proteins in the virus-infected cells are composed of some phosphorylated forms, including a major p37-1 and more phosphorylated minor forms (e.g., p37-2, p37-3, etc.), but little p37-0 is detected (Eriguchi et al., 2002). When the E. coli -produced P protein analogues were incubated with BHK-21 cell lysates, heparin-sensitive phosphorylation occurred as described previously (Takamatsu et al., 1998), giving an additional 40-kDa spot. However, such a p40-like derivative displayed a little more basic pI value than that of the authentic p40 produced in the infected cells; hence, the former was termed p40-0 (pI=4.78), while the latter, p40-1 (pI=4.73). In contrast, p40 produced in the P cDNAtransfected animal cell was detected at the p40-1 position. In addition, staurosporine did not affect the p40-1 production in virus-infected nor the P cDNA-transfected animal cells, while the agent reduced production of hyperphosphorylated forms of p37, resulting in accumulation of p37-1, but not of p37-0. These results suggest that, although p37-0 may become a substrate for the heparin-sensitive protein kinase (PK) in vitro, only p37-1 is a substrate for p40 production catalyzed by heparin-sensitive PK in animal cells, and staurosporine-sensitive PK is involved in the production of more phosphorylated forms of p37, but not in p37-1 production from p37-0.

Perfusion of 99Tcm-labeled CD105 Mab into kidneys from patients with renal carcinoma suggests that CD105 is a promising vascular target.

There is strong published and unpublished evidence that our CD105 Mab E9, which is highly reactive with angiogenic endothelial cells, could be a useful reagent to target the vasculature of solid tumors in man. Since Mab E9 does not cross-react with animal tissues, we undertook here to evaluate its localization using human kidney as an ex vivo model. Perfusion was performed through the renal artery of 99Tcm-labeled purified CD105 Mab in freshly excised kidneys from 7 patients with renal carcinoma. In all 7 cases, immunoscintigraphs showed the presence of well-defined radioactive hot spots, which matched the positions of the tumors as identified by presurgery MRI scans and subsequent histopathologic examination. Importantly, in one instance, where a presurgery MRI scan had identified only one tumor, immunoscintigraphs showed 2 distinct hot spots of radioactivity. The pathology report confirmed that the additional hot spot corresponded to a small secondary well-vascularized tumor. The implication of this finding is that the radiolabeled Mab, E9, may be of use in the detection of metastatic disease. That the labeling of tumors was specific was confirmed when prior perfusion of unlabeled mab E9 in 2 kidneys completely blocked the localization of 99Tcm-conjugated Mab E9. Radioactivity in samples of tumor and normal tissue taken from 7 kidneys was counted in a gamma counter. In all cases, there was a greater uptake of radioactivity in tumors compared with the corresponding normal kidneys. The median values, adjusted per gram wet weight, for 99Tcm were 14.8 times (range, 4.8-113.0) greater in kidney tumors than in normal kidney tissue (p < 0.007). Immunofluorescent staining of cryostat sections of tumor tissues in each of the 7 cases showed strong and uniform localization of Mab E9 in tumor microvessels. Interestingly, chimeric staining of endothelial cells (ECs) was seen in an occasional microvessel segment. That is, while most of the ECs lining a microvessel were strongly stained, an occasional EC was negative. This was not an artifact of staining. Unstained ECs may be nonangiogenic or apoptotic since CD105 is a proliferation/activation-associated antigen. Further investigations are warranted to establish the pharmacokinetics of 99Tcm-labeled CD105 antibody in vivo. This would enable us to determine whether an apparently highly successful ex vivo study has the potential for tumor imaging/therapeutic vascular targeting in patients with cancer.

Identification of α-type subunits of the Xenopus 20S proteasome and analysis of their changes during the meiotic cell cycle

BackgroundThe 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of α4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of α subunits during Xenopus oocyte maturation.ResultsWe identified cDNA for three α-type subunits (α1, α5 and α6) of Xenopus, then prepared antibodies specific for five subunits (α1, α3, α5, α6, and α7). With these antibodies and previously described monoclonal antibodies for subunits α2 and α4, modifications to all α-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except α7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of α2 and α4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase).ConclusionsSix of α-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (α2 and α4) are modified meiotic cell cycle-dependently.

Contribution of lactate buffer, glucose and glucose degradation products to peritoneal injury in vivo.

Long-term peritoneal dialysis (PD) is associated with the development of functional and structural alterations of the peritoneal membrane. In this study, we investigated the contribution of low pH lactate buffer, high glucose concentration and glucose degradation products to peritoneal injury in a rat peritoneal exposure model. Rats received daily 10 ml of either heat-sterilized (3.86% glucose, pH 5.2, n = 8) or filter-sterilized PD fluid (3.86% glucose, pH 5.2, n = 8), or lactate buffer (pH 5.2, n = 8) via a mini vascular access port during a 10 week period. Untreated rats served as controls. The low pH lactate buffer instillation induced pronounced morphological changes including the induction of angiogenesis in various peritoneal tissues and mild damage to the mesothelial cell layer covering the peritoneum. It also evoked a cellular response characterized by an increased mesothelial cell density on the liver, the induction of milky spots and accumulation of omental mast cells in the omentum, and significant changes in the composition of peritoneal leukocytes. The addition of glucose to low pH lactate buffer (filter-sterilized PD fluid) strengthened most, but not all of the responses described above and induced a fibrogenic response. In addition to glucose and low pH lactate buffer, the presence of glucose degradation products (heat-sterilized PD fluid) significantly induced an additional omental milky spot response (P < 0.03) and caused profound mesothelial damage. The vessel density in the omentum and the mesentery was significantly correlated to both the number of tissue mast cells and the hyaluronan content in the peritoneal lavage, which might suggest a role for mast cells and hyaluronan in the induction of angiogenesis. Instillations of low pH lactate buffer, a high glucose concentration and glucose degradation products contribute differently and often cumulatively to peritoneal injury in vivo.

New approach to cancer therapy: the application of signal transduction to anti-cancer drug.

Although it has been widely known that extracellular signal-regulated kinase (ERK) pathway stimulates cell growth and have a protective effect from cell death, recent findings propose pro-apoptotic action of ERK phosphorylation. Since it was found that Vitamin K3 (VK3) or its analog was a potent growth inhibitor and inducer for ERK phosphorylation through specific pathway in cancer cell line, the critical role of ERK phosphorylation in growth inhibitory actions can be discussed. VK3 induced receptor tyrosine phosphorylation and occurred at growth inhibitory concentrations. The phosphorylation of growth factor receptor by VK3 was indicated to be functional, since these were connected with growth factor receptor-bound protein 2 (Grb2) and SOS1. The growth factor stimulated to induce cyclin D1 protein and increase DNA contents. In addition, ERK inhibitor antagonized increase of cyclin D1, suggesting that ERK phosphorylation by growth factor might play an essential role for cell growth. By contrast, ERK phosphorylation by VK3 was more prolonged and intense than the signal induced by growth factors and the antagonize ERK phosphorylation protected from growth inhibition by VK3. The additional and extra ERK spot by VK3, compared with those obtained from growth factor, was detected on two dimensional gels, and this was completely and selectively antagonized by ERK inhibitor. Therefore, the overexpressed ERK phosphorylation was suggested to originate from the additional spot, which played a critical role in growth inhibitory action, despite ERK phosphorylation by growth factor had an essential association with cell growth. The new approach to consider the signal transduction can be one of the most favorite strategies for cancer therapy in the future.

New cercosporoid leaf spot diseases from New Zealand

Several new foliicolous plant pathogens including Pseudocercospora beilschmiediae, P. coprosmae, P. dianellae, P. libertiae, P oleariae, P pomaderridis, Pseudocercosporella helenii, Ramularia coprosmae and Stenella aucklandica are described from New Zealand, and the new combination Pseudocercospora nogalesii is introduced. Furthermore, some additional cercosporoid leaf spot diseases that are new to New Zealand are recorded. The taxonomy of Cercospora on Limonium spp. is discussed and it is shown that two species are involved, viz., Cercospora apii s.lat. (= C. statices Pesante) and C. insulana (= C. statices Lobik).

Generation of free radicals and induction of DNA adducts by activation of heterocyclic aromatic amines via different metabolic pathways in vitro.

Food-derived heterocyclic aromatic amines (HCAs) have proved to be carcinogenic in both rodents and nonhuman primates. Two different metabolic pathways are suggested for the metabolic activation of HCA. The hepatic pathway proceeds via a two-step process involving N-hydroxylation by cytochrome P4501A2 and subsequent O-acetylation by N-acetyltransferase-2. An alternative pathway may be of particular interest in extrahepatic tissues and proceeds via one-electron oxidation catalyzed by prostaglandin H synthase (PHS), rendering free-radical metabolites. In this study, we investigated the metabolic activation of two HCAs, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), by two different enzyme systems in vitro, generating different primary and secondary reactive metabolites. Rat liver S9 mix and PHS were used as the activating system and represent the hepatic and extrahepatic pathways, respectively. Electron-spin resonance spectroscopy showed that both IQ and PhIP exerted inhibiting effects on PHS-mediated formation of hydroxyl radicals during the conversion of arachidonic acid to prostaglandins. Evidence for the formation of HCA free radicals was presented in an indirect way by the formation of glutathione-derived thiyl radicals, with purified PHS as the activating system. Activation by S9 mix did not result in the formation of detectable radical metabolites, showing that the two metabolic routes primarily led to the formation of different metabolites. In all electron-spin resonance experiments, IQ appeared to be more effective than PhIP. In contrasts, DNA adduct analysis by means of (32)P-postlabeling showed similar adduct patterns for S9 and PHS in single-stranded and double-stranded salmon testes DNA after incubation with PhIP, indicating the ultimate formation of a common reactive intermediate. For IQ, activation by PHS led to an additional adduct spot that was not present after S9 activation. Furthermore, activation of IQ resulted in higher adduct levels compared with PhIP for both activation pathways. Overall, adduct levels were higher in single-stranded DNA than double-stranded DNA. Our results showed that the hepatic and extrahepatic pathways resulted in different primary metabolites, while the ultimate formation of a similar reactive intermediate for PhIP, possibly an arylnitrenium ion, suggested that both pathways could play an important role in the onset of carcinogenesis.

Mutations in the RYR1 gene in Italian patients at risk for Malignant Hyperthermia: evidence for a cluster of novel mutations in the C-terminal region

Mutations in the ryanodine receptor type 1 (RYR1) gene are associated with Malignant Hyperthermia (MH) and Central Core Disease (CCD). We report here on the molecular analysis of the RYR1 gene in Italian families referred as potential cases of MH or in patients with CCD or multicore/minicore myopathy. Of a total of 20 individuals with mutations in the RYR1 gene, 14 were part of a group of 47 MH susceptible (MHS) patients, 4 of 34 individuals diagnosed as MH equivocal (MHE), and 2 were patients diagnosed with minicore myopathy and CCD, respectively. Mutations were found to segregate with the MHS or MHE phenotype within the families of the probands. A discordance between phenotype and genotype was observed in a family where a mutation detected in an MHS proband was also found in the father who had been diagnosed MH normal (MHN) at the IVCT. In addition to known mutations, seven novel mutations were found, five of which occurred in exons encoding the C-terminal region of RYR1. These results indicate that the C-terminal region of RYR1 represents an additional hot spot for mutations in patients with MH, similar to what has been reported for patients with CCD.

Identification and Characterization of Mycobacterial Proteins Differentially Expressed under Standing and Shaking Culture Conditions, Including Rv2623 from a Novel Class of Putative ATP-Binding Proteins

The environmental signals that affect gene regulation in Mycobacterium tuberculosis remain largely unknown despite their importance to tuberculosis pathogenesis. Other work has shown that several promoters, including acr (also known as hspX) (alpha-crystallin homolog), are upregulated in shallow standing cultures compared with constantly shaking cultures. Each of these promoters is also induced to a similar extent within macrophages. The present study used two-dimensional gel electrophoresis and mass spectrometry to further characterize differences in mycobacterial protein expression during growth under standing and shaking culture conditions. Metabolic labeling of M. bovis BCG showed that at least 45 proteins were differentially expressed under standing and shaking culture conditions. Rv2623, CysA2-CysA3, Gap, and Acr were identified from each of four spots or gel bands that were specifically increased in bacteria from standing cultures. An additional standing-induced spot contained two comigrating proteins, GlcB and KatG. The greatest induction was observed with Rv2623, a 32-kDa protein of unknown function that was strongly expressed under standing conditions and absent in shaking cultures. Analysis using PROBE, a multiple sequence alignment and database mining tool, classified M. tuberculosis Rv2623 as a member of a novel class of ATP-binding proteins that may be involved in M. tuberculosis's response to environmental signals. These studies demonstrate the power of combined proteomic and computational approaches and demonstrate that subtle differences in bacterial culture conditions may have important implications for the study of gene expression in mycobacteria.

Glutamine synthetase in experimental meningitis: increased ratio of the subunits 3 and 2 may indicate enhanced activity

Glutamine synthetase (GS) activity is higher in the neocortex but not in the hippocampal formation of rabbit brain during Streptococcus pneumoniae meningitis compared to the respective brain region of uninfected control animals. One-dimensional polyacrylamide gel electrophoresis (1D-SDS-PAGE) revealed an apparent molecular mass ( M r) of 44 000 Dalton (Da) for GS from rabbit brain. After two-dimensional gel electrophoresis (2D-PAGE), followed by Coomassie-blue staining, GS separated into three distinct spots (S1, S2, S3). One additional spot (S4) occurred on the immunoblot. All four GS spots exhibited the same M r (44 000 Da), but differed in their isoelectric points. Densitometric evaluation of the two-dimensional maps revealed a strong increase of optical density (OD) of S3 in the frontal cortex of infected animals. The calculated OD ratio S3/S2 in the frontal cortex from rabbits with meningitis was 1.75±0.68 (mean±standard deviation). Compared to controls (0.85±0.39), this value was significantly increased ( p=0.0006). In the hippocampal formation, the ratio S3/S2 was nearly unchanged during meningitis. It is suggested that the ratio S3/S2 may indicate a neuroprotective feature of rabbit brain during meningitis since neuronal apoptosis occurs only in the dentate gyrus and not in the frontal cortex.