Organophosphorus insecticide resistance in a new strain of Culex quinquefasciatus (Diptera: Culicidae) from Tanga, Tanzania

1993 ◽  
Vol 83 (1) ◽  
pp. 67-74 ◽  
Author(s):  
A. Khayrandish ◽  
R.J. Wood
Keyword(s):  
Insecticide Resistance ◽  
Culex Quinquefasciatus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Laboratory Culture ◽  
Fourth Instar ◽  
Resistance Allele ◽  
Susceptible Control ◽  
The Mean

AbstractFourth instar larvae of a new strain of Culex quinquefasciatus Say from Tanzania (TANGA) were tested for insecticide resistance. Initially, the resistance ratio (RR) to chlorpyrifos was 41.8, to temephos 30.8, to propoxur 3.7. After 2–3 years of laboratory culture, resistance to chlorpyrifos and propoxur had declined (chlorpyrifos 5.7, 3.8; propoxur 1.9, permethrin 1.9). Significant synergism was found between s, s, s-tributyl trithiophosphate (DEF) and chlorphyrifos, reducing the RR from 8.0 to 2.5. Synergism between piperonyl butoxide and permethrin was less than in a susceptible control strain. Twelve esterase isozymes of different relative mobilities (Rm) on polyacrylamide gel electrophoresis were identified, ten of which remained when the strain was reinvestigated two years (approximately 32 generations) later. Null activity for all but one of these bands was observed in some larvae. Four esterase bands (Rm 0.25, 0.27, 0.31, 0.34, designated A2, A3, B2, B3) showed polymorphism in activity, with very intense bands in some larvae. The mean frequency of bands with activity greater than standard, declined as organophosphorus (OP) resistance declined, but resistance was unconnected with the frequency of nulls at these positions. In mass larval assays of in vitro sensitivity of acetylcholinesterase (AChE) to propoxur, the I50 exceeded 10x10−4M, compared with 0.1x10−4M in a reverted resistant strain (RANGOON). Single larvel assays revealed heterogeneity, which was interpreted on the basis of an AChE resistance allele (AceR) with a frequency of 0.23.

scholarly journals Purification and properties of an esterase from organophosphate-resistant strain of the mosquito Culex quinquefasciatus

1990 ◽  
Vol 266 (1) ◽  
pp. 83-90 ◽  
Author(s):  
A T Merryweather ◽  
J M Crampton ◽  
H Townson
Keyword(s):  
Culex Quinquefasciatus ◽  
Resistant Strain ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Starch Gel Electrophoresis ◽  
Post Translational Modification ◽  
Highly Active ◽  
High Concentrations ◽  
Starch Gels

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.

A multiple basis for insecticide resistance in a strain of Culex quinquefasciatus (Diptera: Culicidae) from Muheza, Tanzania, studied as resistance declined

1993 ◽  
Vol 83 (1) ◽  
pp. 75-85 ◽  
Author(s):  
A. Khayrandish ◽  
R.J. Wood
Keyword(s):  
Culex Quinquefasciatus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Laboratory Culture ◽  
Dominant Effect ◽  
Level Of Activity ◽  
Standard Level ◽  
Minor Influence ◽  
Initial Resistance ◽  
A Minor

AbstractEvidence from observing resistance in decline in fourth instar larvae of the multiple resistant MUHEZA strain of Culex quinquefasciatus Say from Tanzania (MUHEZA), showed that the major mechanisms for chlorpyrifos and propoxur resistance were different. Resistance to chlorpyrifos declined more than 400-fold (from an initial resistance ratio (RR) of 14285) while resistance to propoxur remained stable for at least 30 generations of laboratory culture. Significant synergism was found between propoxur and piperonyl butoxide (PB), propoxur and s,s,s-tributyl trithiophosphate (DEF), and permethrin and PB, but antagonism occurred between chlorpyrifos and PB, and no synergism between chlorpyrifos and DEF. Nine esterase isozymes active against naphthyl acetates on polyacrylamide gel electrophoresis (PAGE), were identified, four (A2, A3, B2, B3) showing polymorphism in activity, with very intense expression at one or other position in more than 32% of larvae, and null expression in less than 30%. The frequency of intense bands and of nulls both declined as resistance declined. A selected substrain (MUHEZA-fb), breeding true for A2, A3, B2 and B3 at the standard level of activity, showed almost stable chlorpyrifos resistance (RR=1428–1785) for approximately 35 generations. In mass larval assays of in vitro sensitivity of acetylcholinesterase (AChE) to propoxur, the I50 in MUHEZA was 950-fold greater than in a reverted resistant strain (RANGOON). Single larval assays indicated an AChE resistance allele (AceR) at frequency 0.43. PAGE of AChE revealed nine isozymes in MUHEZA and five in RANGOON, three of which were in common. It is concluded that propoxur resistance was due principally to AceR with a minor influence from oxidases and non-specific esterases, while chlorpyrifos resistance was due to an interaction between AceR and non-specific esterases, the latter exerting the dominant effect.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

In-vivo and in-vitro secretion of prolactin by lactating rat adenohypophyses as a function of intracellular age

1984 ◽  
Vol 101 (1) ◽  
pp. 27-32 ◽  
Author(s):  
F. Mena ◽  
G. Martínez-Escalera ◽  
C. Clapp ◽  
C. E. Grosvenor
Keyword(s):  
Pituitary Gland ◽  
Incubation Period ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Pituitary Glands ◽  
H 26

ABSTRACT Adenohypophysial prolactin of lactating rats was pulse-labelled by [3H]leucine injected i.v. at the time of removal of the pups. The [3H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [3H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups. In rats whose pups were removed for 8 h, suckling also depleted–transformed [3H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [3H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [3H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [3H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer. J. Endocr. (1984) 101, 27–32

scholarly journals Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

1982 ◽  
Vol 2 (4) ◽  
pp. 412-425 ◽  
Author(s):  
S I Reed ◽  
J Ferguson ◽  
J C Groppe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Mutagenesis ◽  
Coding Region ◽  
Loop Analysis ◽  
Partial Digestion ◽  
Polyadenylic Acid

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

Monoclonal antibodies against surface antigens of schistosomula of Schistosoma mansoni

Parasitology ◽  
1982 ◽  
Vol 84 (1) ◽  
pp. 65-82 ◽  
Author(s):  
D. W. Taylor ◽  
A. F. Butterworth
Keyword(s):  
Monoclonal Antibodies ◽  
Gel Electrophoresis ◽  
Primary Infection ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Surface Antigens ◽  
Soft Agar ◽  
Surface Proteins ◽  
Spleen Cells

SUMMARYMonoclonal antibodies have been produced after fusion of NS-1 murine myeloma cells with spleen cells from mice immunized either by chronic primary infection or with irradiated cercariae: in both cases, animals were challenged with live cercariae 7 days before fusion. The initial cultures were screened for anti-schistosomular antibodies both by a radioimmunoassay with whole schistosomulum extracts and by immunofluorescence. There was no correlation between the two techniques and subsequent screening was carried out by immunofluorescence. Cloning was carried out in soft agar and 7 cloned cell lines, from 5 initial cultures, were selected for detailed study. Products of 6 of these 7 lines were monoclonal, as judged by isoelectricfocusing of [35S]methionine-labelled supernatant fluids, and their binding to live schistosomula was specific. None of the antibodies showed detectable activity in mediating eosinophil- or complement-dependent damage to schistosomula in vitro. However, 2 antibodies were successfully used to isolate surface proteins with an apparent molecular weight of 24000 on SDS-polyacrylamide gel electrophoresis.

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

Identification of canine pulmonary surfactant-associated glycoprotein A precursors

1985 ◽  
Vol 58 (6) ◽  
pp. 2091-2095 ◽  
Author(s):  
T. E. Weaver ◽  
J. A. Whitsett ◽  
W. M. Hull ◽  
G. Ross
Keyword(s):  
Gel Electrophoresis ◽  
Pulmonary Surfactant ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surfactant Protein ◽  
Lung Lavage ◽  
Complex Carbohydrate ◽  
Monospecific Antiserum ◽  
Microsomal Membranes

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000–28,000 daltons; glycoprotein A2, 32,000–34,000 daltons; and glycoprotein A3, 37,000–38,000 daltons; pH at isoelectric point (pI) 4.5–5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8–5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000–34,000 daltons, pI 4.8–5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000–28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro

1997 ◽  
Vol 9 (6) ◽  
pp. 603 ◽  
Author(s):  
J. C. Bell ◽  
L. C. Smith ◽  
R. Rumpf ◽  
A. K. Goff
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Uv Exposure ◽  
One Dimensional ◽  
Repair Mechanisms ◽  
Bovine Oocytes

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

scholarly journals Effects of some antibiotics on glucose 6-phosphate dehydrogenase in sheep liver

2012 ◽  
Vol 47 (No. 10 - 11) ◽  
pp. 283-288 ◽  
Author(s):  
M. Çiftci ◽  
V. Turkoglu ◽  
S. Aldemir
Keyword(s):  
Gel Electrophoresis ◽  
Enzyme Purification ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sheep Liver ◽  
Sds Page ◽  
In Vitro Effects ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

In vitro effects of penicillin, sulbactam, cefazolin, and amikacine on the activity of the enzyme glucose-6-phosphate dehydrogenase in sheep liver were investigated. Glucose 6-phosphate dehydrogenase was purified from sheep liver, using a simple and rapid method. The purification consisted of two steps, preparation of homogenate and 2’, 5’-ADP Sepharose 4B affinity chromatography. As a result of the two consecutive procedures, the enzyme, having the specific activity of 11.76 EU/mg proteins, was purified with a yield of 35.72% and 1.913 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for the enzyme. In addition, I50 values of the antibiotics were determined by plotting activity % vs. antibiotic concentrations. I50 values were 17.71 mM for penicillin, 27.38 mM for sulbactam, 28.88 mM for cefazolin, and 30.59 mM for amikacine.

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