Isolation and characterization of a cDNA clone encoding a lectin gene from Agaricus bisporus.
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Agaricus↗
Biomedical subjects
M Moyer
Publications and source records attributed to M Moyer.
Effect of K+ channel blockade on fatigue in rat diaphragm muscle.
K+ channel blockers increase skeletal muscle force during twitch contractions; the present study determined whether K+ channel blockade also modulates force during longer term and higher frequency stimulation. 4-Aminopyridine (4-AP; 0.3 mM) increased rat diaphragm force during twitch, 5-Hz and 20-Hz but not 100-Hz stimulation, and prolonged isometric contraction but not half-relaxation time. In response to continuous 5-Hz stimulation, the rate of force decline was accelerated by 4-AP so that over time force dropped below that of control muscle strips. In response to intermittent 20-Hz stimulation, 4-AP produced an early force potentiation; the 4-AP-induced force increase was maintained throughout repetitive stimulation despite an accelerated rate of force decline. In response to continuous 100-Hz stimulation, 4-AP did not affect rate of force decline. During 5- and 20-Hz stimulation, there was an interaction between 4-AP and duration of stimulation in prolonging contraction and especially half-relaxation time. Tetraethylammonium (10 mM) augmented diaphragm force less than did 4-AP, did not affect rate of force decline during 5-Hz stimulation, and did not interact with fatigue to prolong isometric twitch kinetics. These data indicate that K+ channel blockade with 4-AP increases diaphragm force at low to intermediate stimulation frequencies, may increase early force potentiation during repetitive contraction, and depending on stimulation pattern either accelerates or has no effect on rate of fatigue.
4-Aminopyridine↗
Prevention of lateral hip injuries in competitive figure skaters.
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Journal Article↗
Depletion of the erbB-2 gene product p185 by benzoquinoid ansamycins.
Herbimycin A, a benzoquinoid ansamycin, is widely used as an inhibitor of tyrosine kinases. We have examined the effects of herbimycin A and several analogues on p185, the tyrosine kinase encoded by the erbB2 gene in human breast cancer cells. Exposure to 0.35 microM herbimycin A reduced tyrosine phosphorylation of p185 in SKBr3 cells by 80% after 2 h, and the p185 protein level was reduced by 90% after 6 h. The reduction of p185 resulted primarily from increased degradation of p185; cellular protein synthesis was reduced only 16% in SKBr3 cells treated with herbimycin A, RNA synthesis was inhibited only 10%, and erbB2 mRNA levels were unchanged. Examination of the major cellular glycoproteins indicated that most glycoproteins were unaffected under conditions that substantially depleted p185. Studies with cell lines transfected with erbB2 containing defined deletions indicated that susceptibility to the depletion of p185 by herbimycin and its analogues required the domain encoded by amino acids 751-971. The benzoquinoid ansamycins therefore initiate a process of specific degradation of tyrosine kinases by a mechanism that remains unknown.
Anti-Bacterial Agents↗
Inhibition of human and murine cytosolic epoxide hydrolase by group-selective reagents.
1. Human and murine cytosolic epoxide hydrolase were inhibited by thiol-, imidazole- and carboxyl-selective reagents. They were not inhibited by amino-, guanido- or activated serine-selective reagents. 2. Murine, but not human, cytosolic epoxide hydrolase was inhibited by N-bromosuccinimide, a tryptophan selective reagent. 3. Based on sequence data from peptides isolated from CNBr digests, human and murine CEH share areas of sequence homology. Of the five unique human CEH CNBr peptides sequenced, three shared common sequences with one of the unique murine CEH CNBr peptides. The human and murine CEH peptides with common sequences had between 64 and 78% sequence identity. 4. A cysteine important for the activity of murine CEH appears not to be in the active site as judged by N-phenylmaleimide inhibition in the presence and absence of either (2S,3S)-2,3-epoxy-3-(4-nitrophenyl)glycidol, a competitive inhibitor, or trans-stilbene oxide, a substrate.
Amino Acid Sequence↗
Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco.
Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.
Amino Acid Sequence↗
Overexpression of plant histidinol dehydrogenase using a baculovirus expression vector system.
A cDNA encoding cabbage histidinol dehydrogenase, including the chloroplast transit peptide sequence, was overexpressed using a baculovirus expression vector system. The maximum level of the expression of histidinol dehydrogenase was reached 5 days after infection of the insect cells. Two forms of recombinant histidinol dehydrogenase with molecular masses of 53 and 52 kDa, respectively, were obtained by a one-step purification from the cell homogenate. Compared with the 52-kDa form, the 53-kDa form contained 10 additional amino acids at the N-terminus derived from the transit peptide. By incubating the cell homogenate for 2 h at 30 degrees C, the 53-kDa form could be completely converted to the 52-kDa form. This conversion was blocked by leupeptin. Eighty percent of the converted 52-kDa form had Cys at position 31 at the N-terminal amino acid and the rest had Met 33. Kinetic properties of the recombinant enzyme were virtually identical to those of histidinol dehydrogenase isolated from cabbage plants. The overexpression of recombinant cabbage histidinol dehydrogenase in insect cells, the proteolytic processing of the preprotein next to the N-terminus (compared to the mature cabbage enzyme), and its easy purification allow the preparation of large amounts of the active enzyme for structural and functional studies.
Alcohol Oxidoreductases↗
Acquired resistance in Arabidopsis.
Acquired resistance is an important component of the complex disease resistance mechanism in plants, which can result from either pathogen infection or treatment with synthetic, resistance-inducing compounds. In this study, Arabidopsis, a tractable genetic system, is shown to develop resistance to a bacterial and a fungal pathogen following 2,6-dichloroisonicotinic acid (INA) treatment. Three proteins that accumulated to high levels in the apoplast in response to INA treatment were purified and characterized. Expression of the genes corresponding to these proteins was induced by INA, pathogen infection, and salicylic acid, the latter being a putative endogenous signal for acquired resistance. Arabidopsis should serve as a genetic model for studies of this type of immune response in plants.
Amino Acid Sequence↗
Functional duality and structural uniqueness of depressant insect-selective neurotoxins.
Depressant insect-selective neurotoxins derived from scorpion venoms (a) induce in blowfly larvae a short, transient phase of contraction similar to that induced by excitatory neurotoxins followed by a prolonged flaccid paralysis and (b) displace excitatory toxins from their binding sites on insect neuronal membranes. The present study was undertaken in order to examine the basis of these similarities by comparing the primary structures and neuromuscular effects of depressant and excitatory toxins. A new depressant toxin (LqhIT2) was purified from the venom of the Israeli yellow scorpion. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic the effects on the intact animal; i.e., a brief period of repetitive bursts of junction potentials is followed by suppression of their amplitude and finally by a block of neuromuscular transmission. Loose patch clamp recordings indicate that the repetitive activity has a presynaptic origin in the motor nerve and closely resembles the effect of the excitatory toxin AaIT. The final synaptic block is attributed to neuronal membrane depolarization, which results in an increase in spontaneous transmitter release; this effect is not induced by excitatory toxin. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation. The depressant toxins comprise a well-defined family of polypeptides with a high degree of sequence conservation. This group differs considerably in primary structure from the excitatory toxin, with which it shares identical or related binding sites, and from the two groups of scorpion toxins that affect sodium conductance in mammals. The two opposing pharmacological effects of depressant toxins are discussed in light of the above data.
Amino Acid Sequence↗
Two antiviral proteins, gp35 and gp22, correspond to beta-1,3-glucanase and an isoform of PR-5.
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Amino Acid Sequence↗
Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein, Win-1 and Win-2.
The extracellular, acidic pathogenesis-related protein, PR-4, was purified to homogeneity from leaves of Nicotiana tabacum infected with tobacco mosaic virus (TMV) and characterized by partial amino acid sequencing. Complementary DNA clones encoding PR-4 were isolated using an oligonucleotide probe based on the sequence of one of the peptides. The deduced PR-4 protein sequence was found to be related to a family of proteins including hevein and Win-1, which have an amino-terminal lectin domain and a carboxy-terminal domain of unknown function. PR-4 is homologous to the carboxy-terminus of these proteins but does not contain the lectin domain. Thus, the organization of the PR-4 family of proteins is similar to that of the plant chitinase family, in that both contain structural subclasses characterized by the presence or absence of an amino-terminal lectin domain. This observation is consistent with the proposal that the DNA encoding the lectin domain may be capable of transposing to form new genes encoding proteins of more complex, multi-domain structure. The expression of PR-4 mRNA was found to increase dramatically in response to TMV infection and the time course of RNA accumulation was similar to that of other PR proteins.
Amino Acid Sequence↗
HIV seroprevalence survey.
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Acquired Immunodeficiency Syndrome↗
Oral cimetidine improves the accuracy and precision of creatinine clearance in lupus nephritis.
OBJECTIVE: To determine whether short-term use of oral cimetidine improves the precision of creatinine clearance (CCr) and reduces the overestimation of glomerular filtration rate (GFR) that occurs with this test in patients with lupus nephritis (because creatinine is secreted by injured renal tubular cells). DESIGN: Double-blind, placebo-controlled, crossover clinical trial. PATIENTS: Thirteen patients with lupus nephritis with mild renal insufficiency (mean serum creatinine, 230 mumol/L [2.6 mg/dL]; median, 106 mumol/L [1.2 mg/dL]). INTERVENTIONS: Patients were given placebo or cimetidine tablets, 400 mg four times daily for 2 days, with ambulatory 24-hour urine collection during the second 24 hours ("outpatient study"). Simultaneous 4-hour technetium-99-diethylenetriamine penta-acetic acid (Tc99-DTPA) and CCrs were measured immediately after each 24-hour collection ("simultaneous study"). MEASUREMENTS AND MAIN RESULTS: Use of cimetidine improved the accuracy of CCr, as measured by the CDTPA-to-CCr ratio (1.07 [cimetidine] compared with 1.33 [placebo]; P less than 0.05). Cimetidine use also improved the precision of CCr (P less than 0.05). In addition, when compared with standard clinical estimators of GFR, creatinine clearance with cimetidine rendered the most precise estimates of GFR and explained more of the variation in GFR estimation than did any other method (R2 = 0.78 compared with R2 = 0.52 to 0.63). These effects were shown under both simultaneous and outpatient conditions. No side effects due to cimetidine occurred. CONCLUSIONS: In patients with lupus nephritis, the cimetidine-aided CCr offers a compromise between the precise and accurate but expensive and inconvenient research techniques (inulin, iothalamate, or DTPA clearances) and the grossly inaccurate and imprecise but convenient technique (CCr) for determining GFR.
Adult↗
Evidence for a third structural class of beta-1,3-glucanase in tobacco.
Glucan endo-1,3-beta-glucosidases (beta-1,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of beta-1,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a beta-1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized beta-1,3-glucanase. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q'. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of beta-1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.
Amino Acid Sequence↗
Emergence of flat cells from glia in stationary cultures of embryonic chick neural retina.
When embryonic retina is dissociated into a single cell suspension and maintained in stationary culture, a population of flat cells is found on the culture dish. We have carried out a morphologic and immunologic study of the emergence of this population in vitro. Ten- and fourteen-day-old chick embryo retinas were dissociated with trypsin, seeded on glass cover slips for various times, and prepared for scanning electron microscopy (SEM) and immunofluorescence (IF) for Vimentin, an intermediate filament protein. SEM indicates that the characteristic flat cell morphology is initiated in some cells in as little as 30 min after the start of the culture. Not all of the cells that attach flatten. As incubation proceeds, small clusters of cells that had formed in suspension attach to the substrate, and flat cells emerge from them. The flattened cells are positive for Vimentin by IF within 10 min of attachment. The percent of fluorescent cells found on the substrate is constant during the time in culture. This suggests that flat cells do not attach first, followed by neural cells, but that the neural cells and flat cells attach to the dish at the same rate. When aggregates that had formed in suspension attach to the substrate, they are anchored by flat cells that migrate out of the aggregate. Since Vimentin appears in the cultured cells within 10 min, it is unlikely that it has been newly synthesized. Thus, the same cells that contained Vimentin in the retina now express it as flat cells. This supports the hypothesis that flat cells derive from the same cells in the retina that give rise to Müller cells. We have also observed the emergence of a population of cells with short (0.5 micron) microvilli that appear within 8 h of culture. They seem to be a distinct subpopulation of the cells on the upper portion of attached clusters.
Animals↗
Identification of a low molecular weight form of epithelial transforming growth factor.
We have purified a novel form of epithelial transforming growth factor (TGFe) from bovine kidney. Acid ethanol extracts of kidney were fractionated by size exclusion, reverse phase and cation exchange chromatography and activity was monitored by measuring growth of SW13 adrenocortical carcinoma cells in soft agar. The purified material was highly cationic, bound weakly to heparin and gave a band at 13-15000 Mr by SDS-PAGE following Bolton-Hunter iodination. This band correspond to the migration of biological activity extractable from gel slices. The results suggest that we have isolated a truncated form of TGFe which nonetheless retains biological activity.
Animals↗
Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco.
Complementary DNA clones encoding two isoforms of the acidic endochitinase (chitinase, EC 3.2.1.14) from tobacco were isolated. Comparison of amino acid sequences deduced from the cDNA clones and the sequence of peptides derived from purified proteins show that these clones encode the pathogenesis-related proteins PR-P and PR-Q. The cDNA inserts were not homologous to either the bacterial form of chitinase or the form from cucumber but shared significant homology to the basic form of chitinase from tobacco and bean. The acidic isoforms of tobacco chitinase did not contain the amino-terminal, cysteine-rich "hevein" domain found in the basic isoforms, indicating that this domain, which binds chitin, is not essential for chitinolytic activity. The accumulation of mRNA for the pathogenesis-related proteins PR-1, PR-R, PR-P, and PR-Q in Xanthi.nc tobacco leaves following infection with tobacco mosaic virus was measured by primer extension. The results indicate that the induction of these proteins during the local necrotic lesion response to the virus is coordinated at the mRNA level.
Amino Acid Sequence↗
Dimer structure of a neuropeptide precursor established: consequences for processing.
A prohormone (P1) of locust adipokinetic hormone I (AKH I) is shown here to be a homodimer of a 41 residue subunit called the A-chain. The A-chain, from the N terminal, consists of AKH I (10 amino acids starting with pyroglutamate) followed by a Gly-Lys-Arg processing site and then a 28 residues called the alpha chain containing a single cysteine and a potential Arg-Lys processing site. When processed each molecule of the homodimer precursor yields two copies of AKH I and one alpha chain homodimer. We call the alpha-alpha homodimer product of P1 processing AKH precursor related peptide 1 or APRP 1. The Arg-Lys dibasic pair found within the alpha chain is not cleaved in vivo. Our results show that neuropeptide precursors can be dimers and that dimer products can be synthesized by processing of a preformed dimer precursor rather than by dimerization of independent subunits.
Amino Acid Sequence↗