Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.2.2.10 (PNL)
 341 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purposes of this study are to develop an in vivo cell system that is suitable for the immunofluorescent detection of transiently expressed proteins targeted to plant peroxisomes and to determine whether a C-terminal serine-lysine-leucine (SKL) tripeptide, a consensus-targeting signal for mammalian peroxisomes, also targets proteins to plant peroxisomes. Protoplasts from mesophyll cells and from suspension-cultured cells initially were examined for their potential as an in vivo import system. Several were found suitable, but based on a combination of criteria, suspension-cultured tobacco (Nicotiana tabacum L. cv Bright Yellow 2) cells (TBY-2) were chosen. The tobacco cell extracts had catalase activity, and two polypeptides of approximately 55 and 57 kD specifically were detected on immunoblots with anti-cottonseed catalase immunoglobulins G as the probe. Indirect immunofluorescence microscopy with these immunoglobulins G revealed a punctate labeling pattern indicative of endogenous catalase localization within putative TBY-2 peroxisomes. The cells did not have to be completely converted to protoplasts for optimal microscopy; treatment with 0.1% (w/v) pectolyase for 2 h was sufficient. Microprojectile bombardment proved superior for transient transformation of the TBY-2 cells with plasmids encoding beta-glucuronidase, or chloramphenicol acetyltransferase (CAT), or CAT with an added C-terminal tripeptide (CAT-SKL). C-terminal SKL is a consensus, type 1, peroxisome targeting signal. Double indirect immunofluorescent labeling showed that CAT-SKL co-localized with endogenous catalase. Non-punctate, diffuse localization of CAT without SKL provided direct evidence that the C-terminal SKL tripeptide was necessary and sufficient for targeting of CAT to plant peroxisomes. These data demonstrate the effectiveness of this peroxisome targeting signal for plant cells.
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PMID:Development and application of an in vivo plant peroxisome import system. 777 May 24

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnlA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA-); both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried at 5' and 3' truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA- transformants. pnlA- transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.
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PMID:Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnlA. 786 90

99mTc-DMSA renal scintigraphy was utilized to investigate the influence of ESWL on renal function in comparison with that of PNL. In the beginning, the reproducibility of renal uptake rate by the scintigraphy was examined in eleven healthy volunteers under both non-diuretic and diuretic states. The renal uptake rate was shown to be sufficiently reproducible in the same person in the two different trials. However, the differences and the standard deviations were shown to be a few percentages, which were not statistically significant. Changes in the repeated renal uptake rate seem to indicate not only changes of renal function with the treatment but also some technical errors. Herein, to investigate changes in renal function of the therapeutic side, the uptake ratio rate (rate of uptake rate in the therapeutic side/uptake rate in the contral lateral side) was utilized instead of uptake rate. Renal scintigraphy was carried out in 48 patients with unilateral renal stones before and after ESWL or PNL monotherapy or the combined ESWL and PNL therapies. Within one week of treatment, the uptake ratio rate significantly decreased in patients with PNL or the combined ESWL and PNL, although DMSA uptake rate in the therapeutic side did not significantly changes. Neither renal uptake rate nor uptake ratio rate significantly changed after ESWL treatment. There was no significant difference in changes of uptake ratio rate between Siemens Lithostars Plus and the improved Dornier HM-3 lithotriptors. This study indicated that ESWL monotherapy did not affect the uptake ratio rate, although PNL monotherapy and the combined ESWL and PNL therapies may affect the uptake ratio rate to some extent.
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PMID:[Influence of extracorporeal shock wave lithotripsy (ESWL) on renal function assessed by 99mTc-DMSA scintigraphy: comparative analysis between ESWL and percutaneous nephroureterolithotripsy (PNL)]. 786 57

Mucous cells and enteroendocrine cells of the pyloric region of the ruin lizard (Podarcis sicula campestris De Betta) have been examined by lectin histochemical and immunohistochemical methods. Binding to five plant lectins (Canavalia ensiformis, Con A; Triticum vulgare, wheat germ, WGL; Lotus tetragonolobus, winged pea, WPL; Glycine max, soybean, SBL; Arachis hypogaea, peanut, PNL) was performed to characterize glycoconjugates in the secretory products of superficial and glandular mucous cells. Lectin histochemistry revealed the presence of N-acetyl-D-galactosamine, D-galactose and N-acetyl-D-glucosamine in the pyloric superficial cells. Mucous glandular cells mainly contained neutral glycoproteins with terminal residues of galactose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. These cells did not react with Con A after periodate oxidation-sodium borohydride reduction (Paradoxical Con A staining). In the pyloric glands three different types of endocrine cells were identified immunohistochemically: gastrin-, serotonin- and somatostatin-immunoreactive cells; VIP-, bombesin- or cholecystokinin-immunoreactive cells have not been found in the pyloric mucosa.
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PMID:Immunohistochemical investigations on the pyloric glands of the ruin lizard (Podarcis sicula campestris de Betta). 791 80

The purpose of this study was to develop an optimal technique for performing sequential bronchoalveolar lavage (BAL) in a murine animal model. Two general anesthetic regimens and four operative techniques of BAL were tested. Anesthesia by intraperitoneal injection of ketamine hydrochloride (100 mg/kg body wt) resulted in death for four of ten subjects, whereas inhalation of diethyl ether led to death for one of ten subjects. BAL using a balloon catheter under bronchoscopic guidance was comparable with postmortal lavage, tolerated better, and resulted in superior cell retrieval with respect to cell differential (macrophages: 95 +/- 2.3; lymphocytes: 3 +/- 1.2; polymorphonuclear lymphocytes [PNL]: 1.2 +/- 1.4) compared with two other techniques using a bent metal tube/polyethylene tubing combination (macrophages: 19.3 +/- 27.4; lymphocytes: 3.8 +/- 4.3; PNL: 35.5 +/- 35.5) and a bronchoscope/polyethylene tubing combination (macrophages: 11.1 +/- 25.5; lymphocytes: 0.7 +/- 1.0; PNL: 55.8 +/- 41.0). The BAL fluid contained significantly more alveolar macrophages and fewer PNL and epithelial cells (p = 0.0001, p = 0.0025, p = 0.02, respectively). We conclude that the technique using a balloon catheter under bronchoscopic guidance during inhalation of diethyl ether is the procedure of choice and results in a representative sample of BAL.
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PMID:Bronchoalveolar lavage: results of sequential, selective techniques in viable murines. 804 44

Oligodeoxyribonucleotide primers were designed from conserved amino acid (aa) sequences between pectin lyase D (PNLD) from Aspergillus niger and pectate lyases A and E (PELA/E) from Erwinia chrysanthemi. The polymerase chain reaction (PCR) was used with these primers to amplify genomic DNA from the plant pathogenic fungus Glomerella cingulata. Three different 220-bp fragments with homology to PNL-encoding genes from A. niger, and a 320-bp fragment with homology to PEL-encoding genes from Nicotiana tabacum and E. carotovora were cloned. One of the 220-bp PCR products (designated pnlA) was used as a probe to isolate a PNL-encoding gene from a lambda genomic DNA library prepared from G. cingulata. Nucleotide (nt) sequence data revealed that this gene has seven exons and codes for a putative 380-aa protein. The nt sequence of a cDNA clone, prepared using PCR, confirmed the presence of the six introns. The positions of the introns were different from the sites of the five introns present in the three PNL-encoding genes previously sequenced from A. niger. PNLA was synthesised in yeast by cloning the cDNA into the expression vector, pEMBLYex-4, and enzymatically active protein was secreted into the culture medium. Significantly higher expression was achieved when the context of the start codon, CACCATG, was mutated to CAAAATG, a consensus sequence commonly found in highly expressed yeast genes. The produced protein had an isoelectric point (pI) of 9.4, the same as that for the G. cingulata pnlA product.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The pectin lyase-encoding gene (pnl) family from Glomerella cingulata: characterization of pnlA and its expression in yeast. 818 49

A prospective study was undertaken to assess the feasibility and safety of bilateral simultaneous percutaneous nephrolithotomy (BPNL) under single anesthesia. BPNL was attempted in 16 consecutive patients with upper tract urolithiasis suitable for percutaneous treatment bilaterally. Bilateral simultaneous PNL could be accomplished in 14 of 16 cases; the opposite side was abandoned in 2 due to technical reasons. The operating sides could be switched within a short period (15 min) by rotating the patient table by 180 degrees. The average total operating time and irrigation time was 83 and 43 min, respectively. A total of 29 tracts and 18 sessions were required for endourologic treatment of 28 units in 14 patients. There was no significant morbidity. Complete clearance was achieved in 11 of 14 patients; there was insignificant residue in 1, while 2 with major residue required adjunct JJ stenting and extra-corporeal shockwave lithotripsy. The average hospital stay was 5.4 days. After initial proficiency with endourology, preparedness for BPNL is advisable in all such cases.
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PMID:Bilateral simultaneous percutaneous nephrolithotomy. A prospective feasibility study. 852 34

Several mutants of Aspergillus niger, deficient in extracellular protease expression, have been isolated and characterized both genetically and biochemically. The mutant strains, obtained after in vivo UV-mutagenesis of conidiospores and selected by haloscreening on a new dual-substrate plate assay, belong to at least seven different complementation groups. These seven prt loci were assigned to linkage groups using master strains with marked chromosomes. One prt locus (prtC) could be assigned to linkage group I, three (prtB, prtE and prtG) to linkage group III, one (prtF) to linkage group V and the two remaining loci (prtA and prtD) to linkage group VIII. Extracellular proteolytic activities varied from 2 to 3% up to 80% of the protease activity of the parental strain. Assigning the different prt mutants to structural or regulatory genes is difficult since only one structural gene, pepA, has been mapped unambiguously on linkage group I but is not identical to prtC. All prt mutants except for prtC are likely to be regulatory mutants or else belong to a proteolytic cascade because residual activities showed that more proteolytic activities were affected simultaneously. Double mutants were constructed both by recombination and by a second round of mutagenesis. In both cases mutants with further reduced extracellular proteolytic activities were isolated. A sensitive in vitro degradation assay, based on the homologous pectin lyase B (PELB) protein to analyze proteolytic degradation in A. niger, was developed and used to show extremely reduced proteolytic PELB degradation in the culture media of some of these mutants.
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PMID:New protease mutants in Aspergillus niger result in strongly reduced in vitro degradation of target proteins; genetical and biochemical characterization of seven complementation groups. 859 Apr 75

Transgenic filamentous fungi of the species Aspergillus niger, A. nidulans and A. awamori expressing and secreting Erwinia carotovora subsp. atroseptica pectate lyase 3 (PL3) were generated. Correct processing of the pre-enzyme was achieved using the A. niger pectin lyase A (PEL A) signal peptide. With the prepro-peptide of A. niger polygalacturonase II, secreted enzymes still possessed the 6- aa pro-sequence, indicating the importance of the conformation of the precursor protein for correct cleavage of the signal sequence. PL3 expression was markedly increased in media optimized for limited protease activity, and reached 0.4, 0.8 and 2.0 mg/l for expression in A. niger, A. awamori and A. nidulans, respectively. Glycans attached to the PL3 enzymes exhibited species-specific differences, and an increase of molecular mass coincided with reduced specific activities of the enzymes.
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PMID:Expression of an Erwinia pectate lyase in three species of Aspergillus. 862 28

Partially depectinated apple cell walls were digested by pectin lyase or endoglucanase or a combination. By combining these commercial enzymes, a higher yield of 22.2% of carbohydrate material was obtained compared with only 13.9% and 5.7%, respectively, when using them singly. Only small amounts of carbohydrates were extracted by buffer (0.8%). The solubilized extracts were fractionated using a combination of ion-exchange chromatography and gel filtration. The individual subfractions were analysed for neutral sugar and uronic acid content. The results indicated the existence of a synergistic effect between pectin lyase and endoglucanase based on the percentage of material extracted.
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PMID:Enzymatic degradation of cell walls of apples and characterization of solubilized products. 878 36


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