On the two forms of bacteriorhodopsin

In our previous work [(1993) FEBS Lett. 313, 248-250; (1993) Biochem. Int. 30,461-469] M-intermediate formation of wild-type bacteriorhodopsin was shown to involve two components differing in time constants (τ₁ = 60–70 μs and τ₂ = 220–250 μs), which were suggested to reflect two independent pathways of M-intermediate formation. The contribution of the fast M was 4-times higher than the slow one. Our present research on M-intermediate formation in the D115N bacteriorhodopsin mutant revealed the same components but at a contribution ratio of 1:1. Upon lowering the pH, the slow phase of M-formation vanished at a pK of 6.2, and in the pH region 3.0–5.5 only the M-intermediate with a rise time of 60 μs was present. A 5–6 h incubation of D115N bacteriorhodopsin at pH 10.6 resulted in the irreversible transformation of 50% of the protein into a form with a difference absorbance maximum at 460 nm. This form was stable at pH 7.5 and had no photocycle, including M-intermediate formation. The remaining bacteriorhodopsin contained 100% fast M-intermediate. The disappearance of the 250-μs phase concomitant with bR460 formation indicates that at neutral pH bacteriorhodopsin exists as two spectroscopically indistinguishable forms.     

Four fish species new to the Omo-Turkana basin,with comments on the distribution ofNemacheilus abyssinicus (Cypriniformes: Balitoridae) in Ethiopia

Four fish species,Pollimyrus isidori (Mormyridae),Barbus paludinosus, Labeo forskalii (Cyprinidae), andNemacheilus abyssinicus (Balitoridae), new to the Omo-Turkana basin, were recorded from the Gojeb River, a tributary of the Omo River (south-western Ethiopia). Occurrence of the latter species in the upper reaches of the Blue Nile and of the Omo drainage substantiates the belonging of the upper parts of these water systems to the Abyssinian highlands ichthyofaunal province.     

Characterization of a recombinant antibody produced in the course of a high yield fed-batch process

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed. (c) 1994 John Wiley & Sons, Inc.     

Saturated Molecular Map of the Rice Genome Based on an Interspecific Backcross Population

A molecular map has been constructed for the rice genome comprised of 726 markers (mainly restriction fragment length polymorphisms; RFLPs). The mapping population was derived from a backcross between cultivated rice, Oryza sativa, and its wild African relative, Oryza longistaminata. The very high level of polymorphism between these species, combined with the use of polymerase chain reaction-amplified cDNA libraries, contributed to mapping efficiency. A subset of the probes used in this study was previously used to construct an RFLP map derived from an inter subspecific cross, providing a basis for comparison of the two maps and of the relative mapping efficiencies in the two crosses. In addition to the previously described PstI genomic rice library, three cDNA libraries from rice (Oryza), oat (Avena) and barley (Hordeum) were used in this mapping project. Levels of polymorphism detected by each and the frequency of identifying heterologous sequences for use in rice mapping are discussed. Though strong reproductive barriers isolate O. sativa from O. longistaminata, the percentage of markers showing distorted segregation in this backcross population was not significantly different than that observed in an intraspecific F(2) population previously used for mapping. The map contains 1491 cM with an average interval size of 4.0 cM on the framework map, and 2.0 cM overall. A total of 238 markers from the previously described PstI genomic rice library, 250 markers from a cDNA library of rice (Oryza), 112 cDNA markers from oat (Avena), and 20 cDNA markers from a barley (Hordeum) library, two genomic clones from maize (Zea), 11 microsatellite markers, three telomere markers, eleven isozymes, 26 cloned genes, six RAPD, and 47 mutant phenotypes were used in this mapping project. Applications of a molecular map for plant improvement are discussed.     

Regulation of μ-Opioid Receptor mRNA in Rat Globus Pallidus: Effects of Enkephalin Increases Induced by Short- and Long-Term Haloperidol Administration

Abstract: The mRNA encoding μ-opioid receptors is expressed in neurons of the globus pallidus, a region of the basal ganglia that receives a dense enkephalinergic innervation from the striatum. The regulation of the mRNAs encoding the opioid peptide enkephalin in the striatum and the μ-opioid receptor in the globus pallidus was examined with in situ hybridization histochemistry following short- or long-term haloperidol treatments, which alter striatal enkephalin mRNA levels. Animals were administered haloperidol daily for 3 or 7 days (1 mg/kg, s.c.) or continuously for 8 months (1 mg/kg, depot followed by oral). Enkephalin and μ-opioid receptor mRNA levels were unchanged after 3 days of haloperidol treatment. In contrast, the enkephalin mRNA level was increased in the striatum, and μ-opioid receptor mRNA levels were markedly decreased in the globus pallidus after 7 days of haloperidol administration. Similar effects were observed in rats treated with haloperidol for 8 months. The results provide the first evidence of regulation of μ-opioid receptor mRNA in vivo.     

Protein Kinase FA/Glycogen Synthase Kinase-3α After Heparin Potentiation Phosphorylates τ on Sites Abnormally Phosphorylated in Alzheimer''s Disease Brain

In this work, we tested the effect of ion channel blockers and of phorbol ester treatments on [³H]dopamine ([³H]DA) release and neurotensin (NT)-induced facilitation of [³H]DA release from cultures of rat fetal mesencephalic cells. The potassium channel blockers tetraethylammonium and 4-aminopyridine increased basal [³H]DA release and decreased K⁺-evoked [³H]DA release, whereas apamin was without effect. K⁺-evoked [³H]DA release was decreased by ω-conotoxin and nifedipine, totally suppressed by cadmium, and unaffected by amiloride. These results show the differential sensitivity of [³H]DA release to blockade of various ion channels and suggest the involvement of N-type, L-type, and non-L-non-N-type, but not T-type, voltage-sensitive calcium channels in K⁺-evoked release. Phorbol 12-myristate 13-acetate increased both spontaneous and K⁺-evoked [³H]DA release, suggesting a modulatory action of protein kinase C on DA release in this system. Unexpectedly, however, the effects of the phorbol ester were not counteracted by the protein kinase C inhibitors H7, staurosporine, or polymyxin B. NT-induced facilitation of K⁺-evoked [³H]DA release was insensitive to most of the ion channel blockers, except cadmium (64% decrease in NT effect), suggesting that the corresponding potassium' and calcium channels were not involved in the effect of NT on [³H]DA release in this system. The NT effect was totally suppressed by phorbol ester treatments, indicating a possible desensitization of the corresponding transduction mechanisms after protein kinase C activation.     

Studies with Differentially Labeled [11C]Cocaine, [11C]Norcocaine, [11C]Benzoylecgonine,and [11C]-and 4′-[18F]Fluorococaine to Probe the Extent to Which [11C]Cocaine Metabolites Contribute to PET Images of the Baboon Brain

Abstract: The psychostimulant drug of abuse, cocaine (benzoylecgonine methyl ester), is rapidly metabolized by cleavage of its two ester groups, to give benzoylecgonine (BE) and ecgonine methyl ester, and by N-demethylation, to give N-norcocaine (NC). The recent use of [N-methyl-¹¹CH₃]cocaine to image brain cocaine binding sites with positron emission tomography (PET) raises the question of whether PET images partially reflect the distribution and kinetics of labeled cocaine metabolites. We prepared [O-metty/-¹¹CH₃]cocaine by methylation of the sodium salt of BE with [¹¹C]CH₃l, and showed that PET baboon brain scans, as well as regional brain kinetics and plasma time-activity curves corrected for the presence of labeled metabolites, are nearly identical to those seen with [N-methyl-¹¹CH₃]cocaine. This strongly suggests that ¹¹C metabolites do not significantly affect PET images, because the metabolite pattern is different for the two labeled forms of cocaine. In particular, nearly half the ¹¹C in blood plasma at 30 min was [¹¹C]CO₂ when [N-methy/-¹¹CH₃]cocaine was administered, whereas [¹¹C]CO₂ was not formed from [O-methy/-¹¹CH₃]cocaine. Only a trace of [¹¹C]NC was detected in plasma after [O-methyl-¹¹CH₃]cocaine administration. Nearly identical brain PET data were also obtained when 4′-[N-methy/-¹¹CH₃]fluorococaine and 4′-[¹⁸F]fluoro-cocaine (prepared by nucleophilic aromatic substitution from [¹⁸F]fluoride-and 4′-nitrococaine) were compared with [N-methy/-¹¹CH₃]cocaine. In vitro assays with rat brain membranes showed that cocaine and 4′-fluoroco-caine were equipotent at the dopamine reuptake site, but that 4′-fluorococaine was about 100 times more potent at the 5-hydroxytryptamine reuptake site. The studies with positron-emitting 4′-fluorococaines thus support the lack of significance of labeled metabolites or of binding to 5-hydroxytryptamine reuptake sites to PET images taken with [N-methy/-¹¹CH₃]cocaine. [¹¹C]NC prepared by O-methylation of norbenzoylecgonine gave PET images with preferential uptake in striatum, but slower clearance from all brain regions than [O-methy/-¹¹CH₃]cocaine. [¹¹C]BE prepared by N-methylation of norbenzoylecgonine did not show brain uptake.     

Chronic Nicotine Administration Differentially Affects Neurotransmitter Release from Rat Striatal Slices

Abstract: The objective of these experiments was to determine whether the chronic administration of nicotine, at a dose regimen that increases the density of nicotine binding sites, alters the nicotine-induced release of [³H]dopamine ([³H]DA), [³H]norepinephrine ([³H]NE), [³H]serotonin ([³H]5-HT), or [³H]acetylcholine ([³H]ACh) from rat striatal slices. For these experiments, rats received subcutaneous injections of either saline or nicotine bitartrate [1.76 mg (3.6 µmol)/kg, dissolved in saline] twice daily for 10 days, and neurotransmitter release was measured following preloading of the tissues with [³H]DA, [³H]NE, [³H]5-HT, or [³H]choline. Chronic nicotine administration did not affect the accumulation of tritium by striatal slices, the basal release of radioactivity, or the 25 mM KCl-evoked release of neurotransmitter. Superfusion of striatal slices with 1, 10, and 100 µM nicotine increased [³H]DA release in a concentration-dependent manner, and release from slices from nicotine-injected animals was significantly (p < 0.05) greater than release from saline-injected controls; release from the former increased to 132, 191, and 172% of release from the controls following superfusion with 1, 10, and 100 µM nicotine, respectively. Similarly, [³H]5-HT release increased in a concentration-related manner following superfusion with nicotine, and release from slices from nicotine-injected rats was significantly (p < 0.05) greater than that from controls. [³H]5-HT release from slices from nicotine-injected rats evoked by superfusion with 1 and 10 µM nicotine increased to 453 and 217%, respectively, of release from slices from saline-injected animals. The nicotine-induced release of [³H]NE from striatal slices was also concentration dependent but was unaffected by chronic nicotine administration. [³H]ACh release from striatal slices could not be detected when samples were superfused with nicotine but was measurable when tissues were incubated with nicotine. The release of [³H]ACh from slices from nicotine-injected rats was significantly (p < 0.05) less than release from controls and decreased to 36, 83, and 77% of control values following incubation with 1, 10, or 100 µM nicotine, respectively. This decreased [³H]ACh release could not be attributed to methodological differences because slices from nicotine-injected rats incubated with nicotine exhibited an increased [³H]DA release, similar to results from superfusion studies. In addition, it is unlikely that the decreased release of [³H]ACh from striatal slices from nicotine-injected rats was secondary to increased DA release because [³H]ACh release from slices from hippocampus, which is not tonically inhibited by DA, also decreased significantly (p < 0.05) in response to nicotine; hippocampal slices from nicotine-injected rats incubated with 1 and 10 µM nicotine decreased to 42 and 70%, respectively, of release from slices from saline-injected animals. Results indicate that the chronic administration of nicotine increases the ability of nicotine to induce the release of [³H]DA and [³H]5-HT and decreases the ability of nicotine to evoke the release of [³H]ACh but does not alter the nicotine-induced release of [³H]NE from brain slices.     

The dynamics of continuous microbial culture described by cell age distribution and concentration of one substrate

A mathematical model has been considered in which the known equation of McKendrick and Von Foerster for cell age distribution is combined with that for substrate concentration. The dependence of cell division rate on cell age has been taken as a step function. The interrelation between culture parameters describing the substrate consumption and cell division has been found. The shape of cell age distribution as well as the values of substrate and cell concentrations in steady and transient states have been investigated. Stationary regimes at the initial culture state synchronized by ages have been found to be established as damped oscillations and age waves. Under definite conditions the transition from one steady growth regime to another includes sharp single-time age synchronization of the culture.     

Phosphorylation of the duck hepatitis B virus capsid protein associated with conformational changes in the C terminus.

The capsid protein of duck hepatitis B virus (DHBV) is phosphorylated at multiple sites during viral infection. A cluster of sites is located near the C terminus of the 262-amino-acid protein. We have used site-directed mutagenesis to show that three serines and one threonine serve as phosphate acceptor amino acids in the C terminus. An additional six potential phosphate acceptor sites in this region were apparently not utilized. Each serine or threonine that served as a phosphate acceptor was adjacent to a downstream proline, while all six serines that were not acceptors for phosphate residues lacked adjacent downstream prolines. Mutation of the downstream proline to glycine at each site had the same effect as mutating the serine itself, suggesting an SP or TP motif as an essential feature for capsid protein phosphorylation. Phosphorylation at these four sites resulted in complex shifts in electrophoretic mobility in sodium dodecyl sulfate gels of the capsid protein or of a C-terminal peptide containing the phosphorylated sites, suggesting that specific conformations of the C terminus are associated with different combinations of phosphorylated serines. We speculate that distinct functions of the C terminus may be associated with different phosphorylated domains on the intact capsid.