Isoform-Specific Membrane Translocation of Protein Kinase C After Ischemic Preconditioning

Mild cerebral anoxic/ischemic/stress insults promote tolerance and thereby protect the brain from subsequent lethal anoxic/ischemic insults. We examined whether specific activation of PKC , , , or isoforms is associated with ischemic preconditioning (IPC) in rat brain. IPC was produced by a 2-minute global cerebral ischemia. Membrane and cytosolic fractions of the hippocampi were immunoblotted using specific antibodies for PKC, , , and . PKC showed a significant translocation to the membrane fraction from 30 min to 4 h and PKC at 4 h following IPC. In contrast, the membrane/cytosol ratio of PKC showed a tendency to decrease at 30 min and 8 h, and the membrane/cytosol ratio of PKC was significantly decreased from 30 min to 24 h following IPC. These findings indicate PKC isoform-specific membrane translocations in the hippocampus after brief global brain ischemia and suggest that activation of PKC and PKC may be associated with IPC-induced tolerance in the rat hippocampus.     

Degradation of 5-pregnene-3β, 20β-diol by a Nocardia sp.

Summary With growing cells of a Nocardia sp., isolated from soil, the degradation of 5-pregnene-3, 20-diol into 3-[5-oxo-7a-methyl-1 (1-hydroxo)-ethyl-3a-perhydroindane-4]-propionic acid was investigated. The results show that iron is essential for production of the perhydroindanpropionic acid, that this production is greatly enhanced by the presence of calcium and that it is maximal in the pH range 7.0–7.5.Abbreviations used in the text PD 5-pregnene-3, 20-diol (pregnendiol) - PDSA 3-[5-oxo-7a-methyl-1(1-hydroxo)-ethyl-3a-perhydroindane-4]-propionic acid (pregnendiol-secoacid) - PSA 3-[5-oxo-7a-methyl-1-acetyl-3a-perhydroindane-4]-propionic acid (progesterone-secoacid) - EDTA Ethylendiamintetracetic acid - DMSO Dimethylsulfoxide     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

The taxonomic significance of the trunk limbs of the chydoridae (Cladocera)

Summary The differences in the structure of the trunk limbs allow to outline three sections of Chydoridae (see table I and fig. 1), coinciding with the sections distinguished according to the structure of the head pores.

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Chydoridae (Cladocera)

Chydoridae (. ), , .

     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Moraxellae: Identification

According to published reports, all strains ofMoraxella osloensis and Moraxella (Oligella) urethralis grew on a mineral-base medium supplemented with 0.015M (0.123%) sodium acetate, but not all alkalinized a mineral-base medium supplemented with 0.008% bromthymol blue and 0.2% sodium acetate. This seeming discrepancy was examined. The growth of most strains ofM. urethralis was inhibited by 0.008% bromthymol blue, and some by 0.2% anhydrous sodium acetate. All (42) strains ofM. osloensis andM. urethralis alkalinized a mineral-base medium supplemented with 0.1% sodium acetate trihydrate and 0.001% bromthymol blue or phenol red.     

Sodium channel opening as a precursor to inactivation

The time constant of the process producing the delay in Na inactivation development as determined by the two pulse method (_delay) was extracted and compared to that of the slowest Na activation process ₃ for the I _Na during the conditioning pulse of that same determination. _delay and two pulse inactivation _c values were computer generated using a nonlinear least squares algorithm. _h and single pulse inactivation _h values were independently generated for each determination also with the aid of the computer using the same non-linear least squares algorithm. In one determination at 2 mV, _c was 4.68 and _delay 0.494 ms while _h was 4.70 and ₃ 0.491 ms for a _c/_h of 0.996 and a _delay/₃ of 1.006. Mean _delay/₃ from five determinations in four axons, both Cs and K perfused, and spanning a potential range of-27 to 2mV was 1.068. The precursor process to inactivation is channel opening. Some fraction of channels presumably inactivate via another route where prior channel opening is not required.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

Synthesis of N-linked pentasaccharides with isomeric glycosidic linkage

As part of a program to explore the structural requirement of N-glycans in the carbohydrate-mediated biological interactions, N-linked pentasaccharide core structure was stereochemically modified in terms of glycosidic linkage. Three isomers, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, and -D-Man-(13)-[-D-man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, were synthesized. Synthesis of the pentasaccharide with natural linkage is also described.     

Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues

The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca²⁺ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC were included for comparison.The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C , ₁, , , and / were all present in rat midbrain cytosolic extract, PKC , ₁, , and / were present in spleen cytosol, and PKC and / were present in COS 7 cell cytosol. The predominance of and activities in COS 7 and spleen extracts respectively was confirmed by enzymic assay.The PKC activity assay was configured such that the Ca²⁺ dependence of the PKC activity induced by different PKC activators could be determined. Phorbol 12,13-dibutyrate (PDBu) was virtually equipotent on the Ca²⁺-dependent PKC activity from midbrain and spleen and slightly less potent on that from COS 7 cells. In the absence of Ca²⁺, PDBu was considerably less potent overall (as, indeed, were the other PKC activators) and was less potent on COS 7 cell PKC than on those from midbrain or spleen. Mezerein was more potent than PDBu at inducing PKC activity in COS 7 cell extracts in either the absence or presence of Ca²⁺ whereas in the presence of Ca²⁺, mezerein was slightly less potent on midbrain and spleen than PDBu and equipotent in the absence of Ca²⁺. Maximum values for Ca²⁺-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca²⁺-dependent PKC isoform activity in a Ca²⁺ free environment. The greater potency of mezerein on PKC was confirmed using PKC and further purified from rat spleen by hydroxylapatite (HAP) chromatography. The effects of both PDBu and mezerein were investigated using anterior pituitary tissue where a particularly high potency of mezerein in the absence of Ca²⁺ was noted. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG), appeared to cause little or no activation of native Ca²⁺-dependent isoforms in Ca²⁺ free conditions unlike another longer chain diacylglycerol, 1,2-dioleoyl-sn-glycerol. Also DOG activated midbrain PKCs more potently than PKCs from spleen or COS 7 cells (or lung and pituitary tissue) in the presence of Ca²⁺. The concentration-dependence of DOG was examined on PKC and PKC further purified from brain by HAP chromatography, revealing that DOG was equally potent on both of these isoforms derived from brain and on recombinant PKC . However, [³H]PDBu binding data using PKC purified from several sources gave very different IC₅₀ values when DOG was used as a displacer, and in general these values correlated with the EC₅₀ values recorded from the activity assay.The data presented here indicate that there are distinct differences in the activator pharmacology of different native PKC isoforms and between the same isoform expressed in different tissues, either because of post-translational modifications or some other tissue specific factor.     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

Inheritance of callus formation ability in anther cultures of rice,Oryza sativa L.

Summary Inheritance of ability to form callus in rice anther culture was studied using the diallel technique. Anthers containing uninucleate microspores from two japonica cultivais (Minehikari and Taipei 309), two indica cultivars (Mingolo and Suweon 290), and 12 F₁'s of the diallel crosses involving these four parents were cultured on Chaleffs R₂ medium and evaluated for callus induction. The parents showed significant differences in anther callus formation, from 41.9% (Taipei 309) to 0% (Suweon 290). Callus induction ability was inherited as a recessive character conditioned by a single block of genes. Additive gene effects were predominant. The japonica types seemed to be good combiners for callus induction. The order of dominance among the four parents was Suweon 290, Mingolo, Minehikari and Taipei 309.     

The Synthesis and Antiviral Activity of Glycyrrhizic Acid Conjugates with α-D-Glucosamine and Some Glycosylamines

Glycyrrhizic acid and its 30-methyl ester were conjugated with 2-amino-1,3,4,6-tetra-O-acetyl-2-deoxy--D-glucopyranose, 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl amine, 2,3,4-tri-O-acetyl--L-arabinopyranosyl amine, 2-acetamido-2-deoxy--D-glucopyranosyl amine, and -D-galactopyranosyl amine using N,N-dicyclohexylcarbodiimide and its mixtures with N-hydroxybenzotriazole. Structures of the conjugates were confirmed by IR, UV, ¹H, and ¹³C NMR spectroscopy. The glycoconjugate with the residues of 2-acetamido-2-deoxy--D-glucopyranosyl amine in the carbohydrate part of its molecule exhibited antiviral activity (ID₅₀ 4 g/ml) toward the herpes simplex type 1 virus (HSV-1) in the VERO cell culture. Two compounds demonstrated anti-HIV-1 activity (50–70% inhibition of p24) in a culture of MT-4 cells at concentrations of 0.5–20 g/ml.     

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

Summary The fluorescent intercalation complex of ethidium bromide (ETB) with DNA was used as a probe to compare the effects of various radicals with respect to impairment of the DNA base-pair region.OH radicals inhibit up to 0.7 dye intercalations perOH at low salt concentration, and for various oxidizing species the effect decreases in the orderOH > Br ₂ ^– > N ₃ > $$ " align="middle" border="0"> (SCN) ₂ ^–. DNA impairment by theOH product of Met-Gly is comparable to that of N ₃ , but no effect was found due to the interaction between DNA and Lys-Tyr-Lys phenoxyl radicals. The reducing speciese _aq ^– , H, O ₂ ^–, and CO ₂ ^– hardly affect the DNA-ETB intercalation.     

Genetic analysis of brown planthopper resistance in twenty varieties of rice,Oryza saliva L.

Summary The inheritance of resistance to brown planthopper, Nilaparvata lugens (Stol.), of 20 rice cultivars was studied. Single dominant genes that are allelic to Bph 3 condition the resistance in cultivars Ptb 19, Gangala (Acc. 7733), Gangala (Acc. 15207), Horana Mawee, Kuruhondarwala, Mudu Kiriyal and Muthumanikam. Single recessive genes that are allelic to bph 4 govern the resistance in cultivars Gambada Samba, Heenhoranamawee, Hotel Samba, Kahata Samba, Kalukuruwee, Lekam Samba, Senawee, Sulai, Thirissa and Vellai Illankali. The resistance in Ptb 33, Sudu Hondarwala, and Sinna Sivappu is governed by one dominant and one recessive gene which segregate independently of each other. The dominant resistance genes in these cultivars appear allelic to either Bph 1 or Bph 3. Similarly, the recessive genes in these cultivars seem allelic to either bph 2 or bph 4. Further investigations are needed to conclusively determine the allelic relationships of resistance genes in Ptb 33, Sudu Hondarwala and Sinna Sivappu.     

The aerobic nitrogen-fixingSynechococcus RF-1 containing uncommon polyglucan granules and multiple forms of α-amylase

The aerobic nitrogen-fixing unicellular cyanobacteriumSynechococcus RF-1 (pcc 8801) contains numerous irregular polyglucan granules ranging in size from 0.1 to 0.4 m. These morphologically unusual polyglucan granules are not found in the sheathed nitrogen-fixing unicellular cyanobacteriumGloeothece RF-6 (pcc 8803). Two forms of -amylase, ethyleneglycolbis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-sensitive and EGTA-insensitive, were found in theSynechococcus RF-1. In addition to their EGTA sensitivity, the two forms of -amylase also differed in their reactive pH range and in their zymogram disc gel electrophoresis. Each form of -amylase was stable and constant in concentration through a diurnal light/dark cycle.     

Cytological characterization,powdery mildew resistance and storage protein composition of tetraploid and hexaploid 1BL/1RS wheat-rye translocation lines

Summary Progenies of a tetraploid 1BL/1RS wheat-rye translocation line, CV 256, selected from the cross Cando x Veery, were analyzed by means of Giemsa C-banding. CV 256 is cytologically stable for the presence of the 1BL/1RS translocation but still segregating for A- and B-genome chromosomes of Cando and Veery. In CV 256, nucleolar activity of the 1RS NOR locus is suppressed, as judged by the absence of a secondary constriction in that rye segment and the capability of organizing nucleoli. PAGE analysis of prolamins confirmed the presence of two 1RS secalins in all single seeds analyzed. SDS-PAGE analysis of reduced glutenins of single seeds indicated that some seeds contained the Cando Glu-B1 locus (subunits 6+8), some contained the Veery Glu-B1 locus (subunits 7+9) while others contained all four subunits, indicating that the material was heterozygous. Pm8 resistance is expressed in the tetraploid 1BL/1RS translocation line based on the reactions of six well-defined powdery mildew isolates. However, Pm8 resistance is not expressed in the hexaploid wheat cultivars Olymp, Heinrich and Florida, which also contain the 1BL/1RS translocation. Obviously, the existence of the 1BL/1RS translocation is not a proof for the expression of the associated genes. PAGE results did not show a clear linkage between powdery mildew resistance and the presence of 1RS secalins.     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo