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Biomedical subjects

W F Hink

Publications and source records attributed to W F Hink.

At least 19 recordsLinked to original sources
PubMed1992

High-level expression using baculovirus, purification, and characterization of a monomeric form of type II calmodulin-dependent protein kinase.

The type II calmodulin-dependent protein kinase is an oligomer existing in multiple isozymic forms. To facilitate investigations of the regulatory mechanisms of this complex enzyme, we have constructed a truncated, calmodulin-dependent monomer of the alpha subunit. The N-terminal enzyme fragment (alpha 315) was expressed at high levels in a baculovirus/insect cell expression system. The recombinant protein was purified chromatographically using DEAE-cellulose, calmodulin-Sepharose, and AffiGel blue, yielding 4 mg of kinase from 1.5 x 10(8) cells in 4 h. Characterization of the truncated kinase indicated that it is a monomer and that interactions of alpha 315 with calmodulin and substrates are indistinguishable from those observed for purified holoenzyme from rat brain. These results indicate that the baculovirus/insect cell expression system is well suited for producing alpha 315, a structurally simplified model of the type II calmodulin-dependent protein kinase.

Animals↗
PubMed1991

A serum-free medium for the culture of insect cells and production of recombinant proteins.

A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.

Animals↗
PubMed1991

Effect of an experimental systemic compound, CGA-184699, on life stages of the cat flea (Siphonaptera: Pulicidae).

The experimental drug CGA-184699 (N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenylaminoc arbonyl]-2,6-difluorobenzamide) was evaluated for efficacy against cat fleas, Ctenocephalides felis (Bouché), held in flea cages on cats. When administered orally, the compound acts systemically and prevents development of the next generation of fleas. There was no effect on adults, but eggs from adults that fed on treated cats had reduced viability. Most larvae that emerged from surviving eggs died. Within the first 2 wk after treatment of cats with CGA-184699, most death of progeny occurred in the egg stage, but as time passed, more eggs hatched but larval mortality prevented development to adults. A single oral dose of CGA-184699 virtually eliminated the next generation of adult fleas for a period of 44 d.

Animals↗
PubMed1990

Physical mapping and field inversion gel electrophoresis of Amsacta moorei entomopoxvirus DNA.

Agarose in situ digestion was used to prepare intact Amsacta moorei entomopoxvirus (AmEPV) DNA from embedded occlusion bodies (OBs). Direct dissolution of OBs in agarose eliminated the necessity for separate purification of virions. A physical map of AmEPV DNA was constructed for five restriction enzymes (BamHI, EcoRI, HindIII, PstI, and XhoI) using single and multiple digests, and isolated fragment digestions. End fragments were identified by Bal31 digestion and snap-back analysis. A least squares procedure was used to reconcile fragment lengths. AmEPV genome size estimates were based on restriction enzyme (REN) fragment length totals (222 kb), reconciled physical map distance (225 kb), and field inversion gel electrophoresis (FIGE) (about 242 kb). Presumably due to the high A + T content (18.5% G + C) of AmEPV DNA, FIGE values for the intact genome and large REN fragments were about 6 to 10% higher than expected. Preparative FIGE was used to concentrate AmEPV DNA from agarose microbead encapsulated insect cells (Estigmene acrea, BTI-EAA). REN digests of this DNA were identical to those from OBs from caterpillars.

Animals↗
PubMed1989

Inhibition of human platelet aggregation and secretion by ant venom and a compound isolated from venom.

Venom from the tropical ant, Pseudomyrmex triplarinus, has activity against rheumatoid arthritis. Since platelets are involved in inflammatory responses, they were employed to study the effects of venom on prostaglandin-dependent human platelet aggregation and secretion. The assay is very sensitive and uses microliter volumes, which makes it useful as a screen during isolation and characterization of venom components. Whole venom inhibited arachidonic acid- and U46619-induced platelet aggregation with IC50s of 45 and 39 micrograms/ml, respectively. This suggested that venom prevented the action of prostaglandins. Pure venom was fractionated by gel filtration and at least three materials with antiplatelet activity were detected. The smallest component (factor F) was most active and was purified by additional molecular filtration and characterized by UV absorbance, thin-layer chromatography, nuclear magnetic resonance spectra, and activity to platelets. Factor F was identified as adenosine, which is known to stimulate platelet adenylate cyclase and has not been previously reported to be a component of insect venom.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1985

Primary culture of ant venom gland cells.

Venom from the ant Pseudomyrmex triplarinus reduces the symptoms and swelling of rheumatoid arthritis. The cells that produce the venom were dissected from larval and pupal ants and culture conditions studied. Cell dissociation, with minimal amount of damage, was done with 0.25% trypsin at 4 degrees C with subsequent use of soybean trypsin inhibitor. A new medium was formulated and epidermal growth factor, fibroblast growth factor, insulin, cAMP, cGMP, and isoproterenol were beneficial. The optimum osmotic pressure was a relatively high 500 mOSM. Conditioning the medium with an established insect cell line was essential for long-term cell survival. Under these culture conditions the structural and metabolic integrity of the cells were maintained for up to 12 mo.

Animals↗
PubMed1978

Cell cycle analysis and synchronization of the TN-368 insect cell line.

A cell cycle analysis of the Trichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G2, 8.5 hr; M, 0.5 hr; G1, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50 mM thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G2.

Animals↗
PubMed1978

Altered phenotypic expression of ultraviolet-irradiated nuclear polyhedrosis virus.

UV (254 nm) irradiation of nuclear polyhedrosis virus results in formation of small plaques. The small-plaque effect is reversed by photoreactivation. Analysis of plaque formation kinetics indicates a slower formation and subsequent enlargement of plaques from UV-irradiated virus in comparison to non-irradiated virus. A delay in viral DNA synthesis is suggested as an explanation for the observed small-plaque effect.

Animals↗
PubMed1977

A comparison of survival and repair of UV-induced DNA damage in cultured insect versus mammalian cells.

Survival and unscheduled DNA synthesis (UDS) were measured in a cultured insect cell line, TN-368, and a cultured mammalian cell line, V-79-4, following exposure to several fluences of ultraviolet light. TN-368 cells were approximately seven times more resistant to the lethal effects of UV than V-79 cells, as determined by colony formation. The amount of UDS per unit amount of DNA is about the same in both cell types 4 hr after 10-50 J/m2 UV irradiations.

Animals↗