The chemical modification of cysteine-69 of rat liver fatty acid-binding protein (FABP): a fluorescence approach to FABP structure and function

Summary Hepatic-FABP was labelled at cysteine-69 with the fluorescent environmentally sensitive reporter group AEDANS. The labelled protein had an emission maximum at 465 nm indicating that cysteine-69 was buried in a non-polar environment. The modified protein was still able to bind ligands such as oleic acid, oleoyl CoA and haem. The affinity of AEDANS-FABP for haem was unaltered as compared with the native protein indicating that cysteine-69 must be remote from the ligand binding site. The binding of oleic acid did not significantly perturb the fluorescence emission spectrum of the fluorescent reporter group suggesting that there are not large conformational changes in the region of cysteine-69 on fatty acid binding. The binding of stoichiometric amounts of oleoyl CoA was accompanied by a small fluorescence enhancement which suggests that fatty acyl CoAs may interact with other regions of the FABP molecule not involved in fatty acid binding.Abbreviations FABP Fatty Acid-Binding Protein - AEDANS 5-[2-(Acetyl)aminoEthyl]Aminonaphthalene-1-Sulphonic Acid - AAEDANS 5-[2-(Iodoacetyl-AminolEthyl] aminonaphthalene-1-Sulphonic Acid - DTNB 5,5Dithiobis-(2-Nitrobenzoic acid)     

Evidence for inhibitory SH groups in the thiol activated K:Cl cotransporter of low K sheep red blood cells

The monofunctional thiol reagents N-ethylmaleimide (NEM) and methyl methanethiosulfonate (MMTS) stimulate ouabain resistant (OR) electroneutral K:Cl cotransport in LK sheep red blood cells at low, but not at high concentrations. Diamide (DM), on the other hand, only stimulates OR K:Cl flux (Lauf, P.K., J. Memb. Biol. 101: 179–188, 1988). The DM stimulated K:Cl cotransport was decreased toward the control value prior to DM stimulation when NEM or MMTS were added, subsequently. The inhibitory effect was dependent on the compound's concentration and exposure time and, in the case of MMTS, was reversed upon addition of dithiothreitol (DTT). The inhibition was more prominent when NEM treatment was performed at pH 8.0 and disappeared at pH 6.0. In contrast the NEM stimulatory effect was most effective when the pH of NEM treatment was 6.0 (Bauer, J. & Lauf, P.K., J. Memb. Biol. 73: 257–261, 1983). The results suggest the existence of additional, however, inhibitory thiol groups in the already thiol-activated K:Cl cotransporter, with a different pKa value and a lower affinity for NEM or MMTS as compared to the stimulatory thiol groups. Like the activating thiols, the inhibitory sulfhydryls appeared to be inaccessible to non-penetrating thiol reagents and hence, must be located deeper within the red cell membrane.     

Histopathology and cell culture characteristics of liver cells from grc − and grc + rats given diethylnitrosamine

The histopathological response and cell culture characteristics of liver cells from the R16 (grc ^–) strain of rats, which carries an MHC-linked deletion, were examined one week after a single intraperitoneal injection of 200 mg/ kg body weight diethylnitrosamine (DEN) and were compared with the response of liver cells from wild type (grc+) rats. The DEN exposure induced hydropicl vacuolar changes in the parenchymal cells and a limited proliferation of oval cells in the periportal areas of the livers of both grc+ and grc rats. Primary culture of collagenase-digested livers consisted of parenchymal, bile ductular and oval-related cells as determined by cell-specific immunohistochemistry. Subpassaged cells from grc+ rats exhibited oval cell ultrastructural morphology, inducible histochemical staining for gammaglutamyl transpeptidase (GGT), and DEN-associated onset of anchorage-independent growth. Primary cultures of liver cells from R16 rats consistently failed to form cell strains upon subpassage.Abbreviations DEN diethylnitrosamine - grc growth and reproduction complex - GGT gamma-glutamyl transpeptidase - MHC major histocompatibility complex     

Subcutaneous implantable infusion device for chronic intravenous pulsatile gonadotropin-releasing hormone treatment

Preliminary data indicate the potential utility of an implantable subcutaneous device that facilitates chronic intravenous infusion of pulsatile gonadotropin-releasing hormone (GnRH) for ovulation induction. GnRH distribution curves were congruent in control monkeys and those with implanted devices. Tissue tolerance was good in this brief trial. These findings suggest that use of this or a similar implantable device be considered for chronic GnRH administration in human pulse therapy.     

Chemical modification of active sites in relation to the catalytic mechanism of F1

Recent studies of chemically modified F₁-ATPases have provided new information that requires a revision of our thinking on their catalytic mechanism. One of the subunits in F₁-ATPase is distinguishable from the other two both structurally and functionally. The catalytic site and regulatory site of the same subunit are probably sufficiently close to each other, and the interaction between the various catalytic and regulatory sites are probably sufficiently strong to raise the uni-site rate of ATP hydrolysis by several orders of magnitude to that of promoted (multi-site) ATP hydrolysis. Although all three subunits in F₁ possess weak uni-site ATPase activity, only one of them () catalyzes promoted ATP hydrolysis. But all three subunits catalyze ATP synthesis driven by the proton flux. Internal rotation of the ₃₃ or ₃ moiety relative to the remainder of the F₀F₁ complex did not occur during oxidative phosphorylation by reconstituted submitochondrial particles.     

The Agrobacterium tumefaciens T-DNA gene 6b is an onc gene

In this article it is shown that the T-DNA of Agrobacterium tumefaciens contains besides the well-known cyt and aux genes another gene with an oncogenic effect in plants. The gene in question is called 6^b and causes the formation of small tumors in plant species such as Nicotiana glauca and Kalanchoe tubiflora.     

Effect of chloramphenicol and lincomycin on chloroplast DNA amplification in greening pea leaves

The light-induced increase in chloroplast DNA was not inhibited by two inhibitors of protein synthesis on 70S polysomes, chloramphenicol and lincomycin, in greening pea leaves. The changes in chloroplast DNA were observed by fluorescence microscopy and measured by hybridization to specific cloned probes. The results suggest that the light-induced increase in chloroplast DNA proceeds without de novo protein synthesis in the chloroplast, in agreement with those with mutants and cultured leaf tissue.     

Analysis of electric and magnetic fields leaking from induction heaters

Results are presented of an investigation on electric and magnetic fields leaking from inductive (magnetic) heaters that are used for thermal processing of high-power electron tubes and lasers in an industrial plant. Measurements of electric and magnetic fields were done using both commercially available and laboratory-developed instrumentation. Isotropic H-field sensors were developed to allow quantitative evaluation of high-intensity magnetic fields. Ten induction heaters with nominal A.C. power ranging from 2.5 kW to 15 kW and operating at frequencies between 300 kHz and 790 kHz were surveyed. Electric field strengths up to 8 kV/m and magnetic field strengths up to 20 A/m were measured.     

Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

The effects of external electric field on the electron transport system of spinach chloroplasts

Since the thylakoid membranes of an active chloroplast are constantly exposed to the electric fields generated by the electron transport system inside the membranes, we have studied the effects of pretreating chloroplasts of spinach ( Spinacia oleracea L.) leaves with an external AC (alternating current) electric field on their electron transport system. It was found that a few minutes electric field pretreatment (333 V cm^-1 across chloroplast samples), especially at low frequency, irreversibly inhibited the activity of photosystem II (PSII), but under certain conditions, stimulated that of photosystem I (PSI). From the measurements of fluorescence from PSII, we ascribe the inhibition to a lesion close to its reaction center P680, leading to increased dissipation of excitation energy to heat. The effect on PSI was investigated by the reduction of its reaction center, P700 by various artificial donors. We suggest that the stimulative effect can be attributed to a positive shift of the surface charge density of thylakoid membranes that brings about an increase in the accessibility of exogenous electronegative donors.