Analogues of taurine as inhibitors of the phosphorylation of an ≈20K molecular weight protein present in a mitochondrial fraction of the rat retina

Summary It has been previously demonstrated that taurine inhibits the phosphorylation of an 20K apparent molecular weight protein present in the mitochondrial fraction of the rat retina (Lombardini, 1991). In the present studies, various analogues of taurine were tested for their inhibitory activity on the phosphorylation of this 20K protein. The most potent analogues were (±)trans-2-aminocyclopentanesulfonic acid (TAPS) and 1,2,3,4-tetrahydroquinoline-8-sulfonic acid (THQS) which were approximately 21 and 7 times more potent than the parent compound, taurine. Median-effect plots were used to calculate the inhibitory median effect and combination index values for the combined effects of taurine and taurine analogues. From these studies, it was determined that the inhibitory taurine analogues were antagonistic to taurine when used in combination with taurine to inhibit the phosphorylation of the 20K apparent molecular weight protein. It was also concluded that: 1) the distance between the nitrogen and sulfur atoms in the taurine structure was important for inhibitory activity; 2) if the nitrogen atom is either within or attached to an unsaturated ring structure the inhibitory potency was significantly decreased, and 3) if both the sulfur and nitrogen atoms are present within the ring structure the analogue has no activity.     

Identification of a specific protein in the mitochondrial fraction of the rat heart whose phosphorylation is inhibited by taurine

Summary It was previously reported that the mitochondrial fraction of the rat heart contained a specific protein with a molecular weight of approximately 44kDa whose phosphorylation was inhibited by taurine (Lombardini,1994a). Isolation of the 44kDa phosphoprotein on a 1-dimensional polyacrylamide gel using traditional glycine buffers followed by re-electrophoresing the cut out proportion of the gel which corresponds to the 44kDa protein on a tricine-buffered gel resulted in sufficient pure protein for sequence analysis. The results indicate that the 44kDa phosphoprotein is pyruvate dehydrogenase.     

Effects of taurine on the phosphorylation of specific proteins in subcellular fractions of the rat retina

The effects of 20 mM taurine on the phosphorylation of specific proteins in mitochondrial and rod outer segment subcellular fractions of the rat retina were measured. A band of protein with an apparent molecular wieght of 20K was consistently inhibited by taurine. Densitometry measurements performed on gel electrophoresis autoradiograms from the mitochondrial fraction demonstrated a 42.7±8.3% decrease due to taurine (20 mM) in the area corresponding to radioactivity from the 20K phosphoprotein. However, only a 21.2±9.0% decrease was observed due to taurine in the rod outer segment preparation. These data suggest that taurine is exerting its primary effect on the phosphorylation of the 20K molecular weight protein in the mitochondria of the retina. In addition, calmodulin and phorbol ester had no effect on the phosphorylation of the 20K molecular weight protein.     

Foliar CO2photoassimilation and chloroplast linear electron transport rates in nitrogen-sufficient and nitrogen-limited soybean plants

Leaflets of soybean plants which are moderately inorganic nitrogen (N)-limited exhibit either no difference in the rate of net photosynthesis or as much as a 15–23% lower net photosynthesis rate per unit area than leaflets of N-sufficient plants [Robinson JM (1996) Photosynth Res 50: 133–148; Robinson JM (1997a) Int J Plant Sci 158: 32–43]. However, mature leaflets of N-limited soybean plants have a higher CO₂photoassimilation rate per unit chlorophyll than leaflets of N-sufficient soybean plants at both moderate light intensity (500 µmol m^-2s^-1) and saturating light intensity (1200 µmol m^-2s^-1) [Robinson JM (1996) Photosynth Res 50: 133–148]. This study was undertaken to determine whether chloroplast thylakoids isolated from the leaflets of nitrogen-limited soybean plants displayed similar or higher linear electron transport rates (H₂O ferredoxin NADP) per unit chlorophyll than thylakoids isolated from leaflets of N-sufficient plants. Chlorophyll concentration in reaction mixtures containing chloroplast thylakoids prepared from leaflets of N-limited plants was manipulated so that it was similar to the chlorophyll concentration in reaction mixtures of thylakoids prepared from leaflets of N-sufficient plants. Measurements of ferredoxin dependent, NADP dependent, O₂photo-evolution in thylakoid isolates were carried out in saturating light (1500 µmol m^-2s^-1) and with (an uncoupler) in the chloroplast reaction mixtures. Chloroplast thylakoids isolated from N-limited soybean plant leaflets routinely had a 1.5 to 1.7 times higher rate of uncoupled, whole chain electron transport per unit chlorophyll in saturating light than did chloroplast thylakoids isolated from leaflets of N-sufficient plants. The results suggest that the photosystems and photosynthetic electron transport chain components are more active per unit Chl in leaflet chloroplast thylakoids of N-limited soybean plants than in thylakoids of N-sufficient plants.     

Free amino compounds and cell volume regulation in erythrocytes from different marine fish species under hypoosmotic conditions: the role of a taurine channel

Erythrocytes from the marine fish species ballan wrasse (Labrus berggylta Ascanius), bullhead (Myoxocephalus scorpius L.), cod (Gadus morhua L.), dab (Limanda limanda L.), eelpout (Zoarces viviparus L.), flounder (Platichthys flesus L.), lumpfish (Cyclopterus lumpus L.), plaice (Pleuronectes platessa L.), sole (Solea solea L.) and turbot (Scophthalmus maxima L.) possess the capacity for regulatory volume decrease. This property was demonstrated in vitro by reduction of the osmolality of the incubation medium from 330 to 255 mosmol·kg^-1. During the 4-h incubation period only the lumpfish cells completely regained the original volume. Twenty-seven free amino compounds were present in detectable amounts in the erythrocytes. At normal osmolality the taurine content was between 14.0 mol·g dry weight^-1 (lumpfish) and 147.4 mol·g dry weight^-1 (sole). Except in the bullhead, taurine was the quantitatively dominating amino compound in the erythrocytes from all species, and accounted for betwee 23% (lumpfish) and 88% (sole) of the total content of free amino compounds. In each species the regulatory volume decrease was associated with a reduction in the cellular content of taurine. Taurine contributed to between 6% (lumpfish) and 36% (flounder) of the cell shrinkage. There was a significant negative correlation, however, between the cellular concentration of taurine at normal osmolality and the capacity of the cells for regulatory volume decrease. Gamma-aminobutyric acid and/or glycine also contributed to the process of volume regulation, but to a lesser extent than taurine. The volume regulatory efflux of taurine and -aminobutyric acid were mediated by taurine channels. It is suggested that these channels also mediated the reduction in the cellular contents of glycine.Abbreviations cmp counts per minute - dw dry weight - GABA -amino-n-butyric acid - MW molecular weight - SD standard deviation - ww wet weight     

Isolation and partial characterization of a protein kinase NII from wheat germ chromatin

A protein kinase, type NII, has been purified from wheat germ chromatin. The enzyme, which uses both ATP and GTP as phosphoryl donors, catalyzes the phosphorylation of casein, phosvitin and E. coli RNA polymerase, but not of histone proteins. Polypeptide bands at 46 kDa, 37 kDa and 25 kDa were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autophosphorylation of the 25 kDa subunit was observed following incubation of the purified kinase with (-³²P)ATP and (-³²P)GTP.     

Net taurine transport and its inhibition by a taurine antagonist

P₂-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [³H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using⁸⁶Rb⁺ as a marker for intracellular K⁺. Taurine was synthesized in the P₂-fraction when incubated in taurine free medium. At external taurine concentrations below 370 M a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P₂-fraction was achieved at external taurine concentrations exceeding 370 M. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [³H]taurine transport into the P₂-fraction. As the external concentration of taurine was increased, the accumulation of⁸⁶Rb⁺ into the P₂-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.     

Sequential fractionation of sediment phosphate

By means of sequential extractions with Ca-NTA and EDTA, a separation was performed between Fe(OOH) P and CaC0₃P in a few sediments; the remaining fraction, considered to be organic phosphate, was quantified as well. We found that with the commonly used method of extraction with NaOH and H₂S0₄, less Fe(OOH) P and much more CaC0₃ P was found than with the chelating extractants. The organic phosphate pool in live and dead algal material and in some mud samples was partly hydrolysed and therefore recovered as inorganic phosphates with classical extractions. The difference between chelating extractants and the classical ones is discussed.Abbreviations o-P: ortho phosphate (or its concentration) - org-P: organic phosphate - extr-P: extractable sediment bound phosphate - extr-Fe: extractable sediment bound iron - Fe(OOH) P: iron bound, sediment phosphate - CaCO₃ P: calcium bound, sediment phosphate - org-C: organic sediment bound carbon     

High-affinity folate binding in human prostate

Binding of³H-folate in Triton X-100 solubilized human prostate homogenate was of a high-affinity type and displayed apparent positive cooperativity typical of specific folate binding. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. Gel chromatography reveled two major folate binding proteins (M_r100 and 25kDa), but only one single band (M_r65–70 kDa) was detectable on SDS-PAGE and immunoblotting with rabbit-anti human milk folate binding protein. Concentration of folate binding protein in prostate homogenate expressed as maximum³H-folate binding was 1.10 nmol/g protein, and the cross-reactivity with rabbit-anti human milk folate binding protein serum was 15% as determined by an enzyme-linked immunosorbent assay (median values; n=6).     

High-affinity taurine uptake in developing retina

A specific system for taurine transport is present at the early stages of development in both chick and rat retinas. The results obtained with taurine analogs indicate a high degree of specificity of taurine uptake. Two transport systems were detected for the adult rat retina: a high-affinity (K _m 21 M) and a low-affinity transport system (K _m 312 M). On the other hand, in the adult chick retina, only a low-affinity transport system (K _m 580 M) could be detected. Nevertheless, embryo chick retina accumulated [³H]taurine by two different kinetic mechanisms withK _m s of 242 M and 21 M for the low- and high-affinity processes, respectively. Taurine uptake systems were absolutely Na⁺ dependent. The sodium-dependence curve for taurine uptake was sigmoid. These mechanisms appear not to be mediated by a Na⁺ cotransport system. In spite of the differences observed in taurine uptake in both species, in each of them it closely parallels the changes brought about by the morphological and functional maturation of the retina.     

Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Circadian changes in protein-synthesis rate and protein phosphorylation in cell-free extracts of Gonyaulax polyedra

The polysomal pattern of the dinoflagellate Gonyaulax polyedra, cultured under constant conditions, demonstrates a circadian rhythm. The relative amount of polysomes increases during the phase corresponding to the previous night period (=subjective night phase) when the rate of protein synthesis reaches its maximum (Cornelius et al., 1985, Planta 160, 365–370). Cell-free extracts were isolated at different circadian phase. The rate of protein synthesis in the extracts changed rhythmically in the same manner as the rate of protein synthesis in vivo. Substances in the postribosomal supernatants influenced the protein-synthesis rate of the cell-free system, depending on the phase when they were isolated: night factors stimulated protein synthesis in day extracts whereas day factors inhibited protein synthesis in night extracts. These effects were abolished by heating the postribosomal supernatant. In-vitro phosphorylation in parallel probes showed changes in the pattern of phosphorylated proteins. Phosphorylation of one of the proteins (95 kDa) was decreased after addition of night factor(s) and increased after addition of day factor(s). Cyclic-AMP enhanced the rates of protein synthesis and phosphorylation in the day extracts.Abbreviations cAMP cyclic-AMP - CT circadian time - D (N) subjective day (night)phase     

Cell cycle dependent distribution of phosphorylated proteins in microspores and pollen ofBrassica napus L., detected by the monoclonal antibody MPM-2

Summary The monoclonal antibody MPM-2, which interacts with a mitosis-specific phosphorylated epitope, has been used to study phosphorylation of proteins in microspores and pollen ofBrassica napus. One- (1-D) and two-dimensional (2-D) immunoblots revealed that MPM-2 recognized a family of phosphorylated proteins in freshly isolated microspores and pollen. The same set of phosphorylated proteins was found after 8 h of culture at embryogenie (32 °C) and non-embryogenic (18 °C) conditions. Two major spots were observed on 2-D immunoblots, one of which (M_r75 kDa, pI5.1) co-localized with the 70 kDa heat shock protein. Immunolabelling of sectioned microspores and pollen showed that MPM-2 reactive epitopes were predominantly observed in the nucleoplasm from G₁ until G2-phase, and in the cytoplasm during mitosis. This may be due to a cell cycle related translocation of phosphoproteins from the nucleus to the cytoplasm, or alternate phosphorylation and dephosphorylation in nucleus and cytoplasm. Detectability of epitopes on sections depended on the embedding procedure. Cryo processing revealed epitope reactivity in all stages of the cell cycle whereas polyethylene glycol embedded material showed no labelling in the cytoplasm during mitosis. Processing might reduce the antigenicity of cytoplasmic MPM-2 detectable proteins, probably due to dephosphorylation. The MPM-2 detectable epitope was observed in all cells investigated, irrespective of culture conditions, and its intracellular distribution depended on the cell cycle stage and was not related to the developmental fate of the microspores and pollen.     

Effects of culture conditions on taurine uptake by various variants of human endometrial carcinoma cells in culture

Summary. The general properties of the taurine uptake in human endometrial tumoral Ishikawa cells were similar to those usually found in other tissues. Uptake was notably affected by the oxygen pressure, being higher at the physiological pO₂ of the endometrium (40mm Hg, equivalent to 5% O₂) compared to that used under standard experimental culture conditions (160mm Hg or 20% O₂). Uptake of taurine was also density-dependent in Ishikawa cells and was significantly decreased at confluence. Uptake regulation by PKC driven phosphorylation occurs only in growing cells and not in resting cells. The taurine uptake of three Ishikawa cell lines was very different. The taurine uptake of one of the cell lines was affected by estradiol, probably through a non-genomic pathway, whereas tamoxifen had no effect in all cell lines.     

Characterization of the Human Gonadotropin-Releasing Hormone Receptor Heterologously Produced Using the Baculovirus/Insect Cell and the Semliki Forest Virus Systems

1. Two eukaryotic viral systems, the baculovirus/insect cell and the Semliki Forest virus systems, were tested for heterologous expression of human gonadotropin-releasing hormone receptor (GnRHR) cDNA.2. An unmodified as well as a c-myc epitope-tagged human GnRH receptor was produced in two insect cell lines (Spodoptera frugiperda, Trichoplusia ni) after infection with the respective recombinant baculoviruses. In both insect cell lines, the receptor was identified by immunoblot analysis as a triplet of bands between 35 and 40 kDa. After deglycosylation of the receptor the molecular mass decreased to 35 kDa. The GnRH receptor was localized in membrane compartments within the infected insect cells. However, only in membranes of infected Trichoplusia ni insect cells could 2000 receptors per cell be detected.3. Production of the GnRH receptor in BHK cells using the Semliki Forest virus system resulted in 50,000 receptors per cell. A maximal yield of 0.42 pmol/mg membrane protein was obtained 24 hr after electroporation of BHK cells with in vitro synthesized RNA. Binding of the antagonist [¹²⁵I]Cetrorelix was saturable with a K _D of 1.3 nM. The receptor produced in the BHK cells was further characterized by ligand displacement studies. The rank order of agonist and antagonist affinities was Cetrorelix > Triptorelin > Antide > GnRH.     

Cytoskeletal-associated protein kinase and phosphatase activities from cerebral cortex of young rats

We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 M cyclic AMP (cAMP), 1 mM calcium and 1 M calmodulin (Ca²⁺/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 M phorbol 12,13-dibutyrate (Ca²⁺/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca²⁺/calmodulin, respectively, but was unaffected in the presence of Ca²⁺/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 M okadaic acid and 0.01 M microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.     

Partial characterization of neural-inducing factors from Xenopus gastrulae Evidence for a larger protein complex containing the factor

Summary High (Mr 90–110 kDa) and low (Mr 15–30 kDa) molecular weight forms of neural-inducing factors have been found in the supernatant of Xenopus gastrula homogenate. The factors, which are protein in nature, have been partially purified by size exclusion high-performance liquid chromatography (HPLC) and sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The factor of smaller size, which could be derived from a precursor, is associated with other proteins in a larger complex. The neural-inducing factors are not irreversibly inactivated after chemical deglycosylation with trifluoromethansulfonic acid. The neural-inducing protein which is found in ribonucleoprotein (RNP)-particles was partially purified by hydrophobic chromatography. Possible relationships of the factors in different subcellular fractions and their physiological significance are discussed. Offprint requests to: H. Tiedemann     

Colocalization of retinal dystrophin and actin in postsynaptic dendrites of rod and cone photoreceptor synapses

In this paper we demonstrate immunostaining specific for dystrophin in photoreceptor synapses of human, bovine and rat retinas. Cryosections of retinas incubated with dystrophin-specific monoclonal antibodies displayed a punctuate staining pattern in the outer plexiform layer. This pattern resulted from binding of the antibodies to synaptic complexes of both rods and cones, shown by double-labelling with antibodies to either synaptophysin or actin. Confocal laser fluorescence microscopy demonstrated that dystrophin staining colocalized predominantly with actin, which is concentrated in the postsynaptic portions of the synaptic complex. No significant dystrophin immunolabel was seen in the presynaptic terminals labelled with antibodies to synaptophysin, a marker of synaptic vesicles. Immunoblot analysis confirmed the presence of 420 kDa and 360 kDa dystrophin-like polypeptide bands associated with membranes of the bovine retina. We speculate that retinal dystrophin is involved in the linkage of actin filaments to the postsynaptic plasma membrane. Such a linkage may be important for the generation of synaptic microdomains and for certain phenomena of synaptic plasticity. The absence of dystrophin in patients suffering from Duchenne's muscular dystrophy is accompanied by visual problems and abnormalities of the electroretinogram. Therefore it is likely that retinal dystrophin plays a role in certain stages of synaptic transmission between photoreceptors and the postsynaptic dendritic complex formed by horizontal and bipolar cells.     

Performance and seasonal abundance of the neotropical brown stink bug, shape Euschistus heros nymphs and adults on a novel food plant (pigeonpea) and soybean

Pigeonpea, Cajanus cajan (L.) Millsp. (Leguminosae) was found as a novel food plant of the neotropical brown stink bug, Euschistus heros (F.), in Paraná State, Brazil. Laboratory studies indicated nymphs performing better when fed on immature pods of soybean, Glycine max (L.) Merrill (Leguminosae), than on immature pods of pigeonpea. Female nymphs required 1.7 d less to complete development and tended to have a lower mortality on soybean (17.5%) than on pigeonpea (25.0%). At emergence, fresh body weight was greater on soybean than on pigeonpea. Adult E. heros performed better on soybean than on pigeonpea. After 120 d, 35% of adults were alive on soybean and 30% on pigeonpea; 93.3% of females oviposited on soybean, and 73.3% on pigeonpea, with an oviposition delay of 17 days on pigeonpea compared to soybean. Fecundity was 63% greater on soybean than on pigeonpea, and adult body weight gain for females and males at different ages was 6–36% greater on soybean than on pigeonpea. E. heros colonized soybean during January–March, from the pod-setting and filling stages (R3–R6) to maturity (R7–R8), and pigeonpea during April–June. These results indicate that E. heros has expanded its host range to pigeonpea in Brazil; pods of this plant are suitable food, and can support the insect during autumn-early winter, after soybean harvest, and before it enters into partial hibernation.     

Neural network designs for poly- β-hydroxybutyrate production optimization under simulated industrial conditions

Improvement of the fermentation efficiency of poly--hydroxybutyrate (PHB) may make it competitive with chemically synthesized petroleum-based polymers. One step toward this is optimization of fluid dispersion and the feed rates to a fed-batch bioreactor. In a recent study using a fermentation model, dispersion corresponding to a Peclet number of 20 was shown to maximize the productivity of PHB. Here further improvement has been investigated using neural optimization. A comparison of seven neural topologies has shown that while feed-forward and radial basis neural networks are computationally efficient, recurrent networks generate higher concentrations of PHB. All networks enhanced the productivity by 16–93% over model-based optimization.