Polymorphisms and diversity of T-cell receptor-γ proteins expressed in mouse spleen

Bulk populations of T-cell receptor (Tcr) -expressing splenocytes from different inbred strains of mice were examined for the diversity of Tcr proteins. Immunoprecipitations with anti-C1/2, anti-C4, and anti-V1 sera demonstrated that splenocytes from B10.BR, C57BL/6, and C57L strains of mice expressed the same array of Tcr proteins, namely V1-C2, V1-C4, and V2-C1, although the Tcr heterodimers observed for each of these strains were biochemically distinct. Examination of bulk splenic Tcr heterodimers from several other inbred strains of mice demonstrated that each of the strains could be categorized into one of three basic phenotypes. For several reasons, the differences observed between the strains appeared to be solely dependent on polymorphisms of the Tcrg loci. First, F₁ mice co-expressed both parental Tcr phenotypes. Second, the distinguishing polymorphism between mice of phenotype 1 and phenotypes 2 or 3 was due to the presence of an N-linked glycosylation site within the Tcrg-C1 gene segment, previously described for BALB.B and C57BL/6 Tcrg-C1 genes. Finally, the V1-C4 polymorphism between mice of phenotype 3 and phenotypes 1 or 2 was due to differences in core protein size. Furthermore, the three defined Tcr chains were expressed independently of the major histocompatibility complex (MHC) haplotype. Although no striking qualitative differences in Tcr heterodimers were observed between strains (including those with autoimmune disorders), a quantitative difference in the relative amount of C4-encoded proteins was observed on Tcr splenocytes from both newborn euthymic and adult athymic mice when compared to adult Tcr splenocytes from euthymic mice. These results demonstrate that genetic polymorphisms exist among different mouse strains and suggest that selective developmental pressures may govern Tcr expression. Offprint requests to: J. A. Bluestone     

Coccidia of Brazilian edentates: Eimeria cyclopei n.sp. from the silky anteater,Cyclopes didactylus (Linn.) and Eimeria choloepi n.sp. from the two-toed sloth,Choloepus didactylus (Linn.)

Summary Eimeria cyclopei n.sp. is described from the silky anteater, Cyclopes didactylus, from Pará State, north Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in seven days at 26 to 28°C. Oocysts are ellipsoidal to sub-spherical, with a mean size of 28.1 × 23.6 m: the wall is 1.5 to 2.0 m thick, apparently with an outer thin, colourless membrane and two inner, thicker, striated and yellowish layers. There is no micropyle, oocyst residuum or polar body. The mean measurements of sporocysts are 19.0 × 9.0 m, and they are slightly asymmetrical, elongate pear-shape, with a plug-shaped Steida body projecting beyond the end of the sporocyst. Sporozoites are as long as or longer than the sporocysts: The sporocyst residuum is scattered between sporozoites in younger specimens and becomes condensed into rounded mass in older ones. The endogenous stages occur in the epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Uninucleate meront, microgamont and macrogamont precursors are recognizable morphologically. Mature meronts are 20.0 × 15.7 m some produce 12 to 20 merozoites which are 8.7 × 2.0 m, and others 10 to 26 merozoites which are 11.4 × 2.0 to 15.0 × 3.0 m. Mature microgamonts which are 27.5 × 24.1 m, produce from 150 to 170 microgametes of 7.1 × 1.0 m: microgametes have two flagella of unequal length. Mature macrogamonts are 28.4 × 24.5 m Eimeria choloepi n.sp. is recorded from the two-toed sloth, Choloepus didactylus, from the same area of Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in 23 days at 26 to 28°C. Oocysts with a mean size of 23.0 × 20.3 m, have a wall 2.0 to 2.5 m thick which is composed of two thick, yellowish and striated outer layers and a delicate, colourless inner one. There is no micropyle, oocyst residuum or polar granule. Mature sporocysts with a mean size of 11.3 × 7.1 m, are ellipsoidal to egg-shaped and have a poorly developed Steida body. The sporocyst residuum is composed of a small number of large globules: The sporozoites are longer than the sporocyst and strongly recurved. The endogenous stages occur in epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Dimorphic meronts produce 8 to 18 merozoites which are either 13.0 × 2.0 m or 13.0 × 3.0 m. Microgamonts produce 50 to 80 microgametes of 8.0 × 1.0 m. Mature macrogamonts are 18.3 × 17.9 m. ac]19820212     

Geochemical controls for Al, Fe, Mn, Cd, Cu, Pb, and Zn during experimental acidification and recovery of Little Rock Lake, WI, USA

Concentrations of Al, Fe, Mn, Cd, Cu, Pb, and Zn were measured in thereference and treatment basins of Little Rock Lake (Vilas County, Wisconsin), alow-alkalinity, seepage system (pH 6.1, alkalinity25eq/L) during six years of a whole-basinacidificationand the first four years of the lake's recovery. The treatment basin wasacidified with H₂SO₄ in three two-year steps to pH5.6, 5.1, and 4.7. By the end of year 4 of recovery, treatmentbasin pH increased to 5.3 as a result of internal alkalinity generation.During acidification, dissolved Mn and Fe (0.4mpore-size filters) increased at pH 5.6; dissolved Al, Cd, and Zn becameelevated at pH 5.1; and dissolved Pb at pH 4.7. Dissolved Cu remainedsimilar in both basins to pH 4.7. Al, Fe and Mn levels declinedsignificantly during the recovery period, approaching values at pH 5.3intermediate between the concentrations at pH 5.6 and 5.1 during acidification.Dissolved Al and Fe in the reference basin were near the equilibrium levels forsolubility of gibbsite (Al(OH)₃) and amorphousFe(OH)₃(s).The acidified basin was undersaturated relative to gibbsite, and dissolved Alwas limited by pH disequilibrium between the water column and sediments andpossibly by Al-DOC precipitation. Dissolved Fe apparently was controlled bysolubility of amorphous Fe(OH)₃(s) and Fe-DOC precipitation.Dissolved Mn levels in both basins were consistent with manganite[-MnOOH(s)] solubility. Elevated levels of Cd, Pb, and Zn in thetreatment basin during acidification probably resulted from less efficientscavenging of atmospherically-deposited Cd, Pb, and Zn by settling particles.     

Selection of microorganisms which produce raw-starch degrading enzymes

Summary Microorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, -amylase resistant starch (-RS), which is insoluble in water and hardly digested with Bacillus amyloliquefaciens -amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced -amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced -amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.     

Coral growth in high-nutrient,low-pH seawater: a case study of corals cultured at the Waikiki Aquarium,Honolulu, Hawaii

Fifty-seven species of hermatypic corals have been maintained and grown in high-nutrient seawater at the Waikiki Aquarium, Honolulu, Hawaii. In this study we document the chemical conditions of aquarium water in terms of dissolved nutrients and carbon. Aquarium water is characterized by concentrations of inorganic nutrients that are high relative to most natural reef ecosystems: SiO₃ 200 M; PO₄ 0.6 M; NO₃ 5 M; NH₄ 2 M. In contrast, concentrations of organic nutrients are lower than most tropical surface ocean waters: DOP 0.1 M and DON 4 M. The incoming well-water servicing the facility has low pH, crating over-saturation of carbon dioxide. The coral communities in aquaria took up inorganic nutrients and released organic nutrients. Rates of nutrient uptake into aquaria coral communities were similar to nutrient uptake by natural reef communities. Coral growth rates were near the upper rates reported from the field, demonstrating corals can and do flourish in relatively high-nutrient water. The growth of corals does not appear to be inhibited at concentrations of nitrogen up to 5 M. Statements implying that corals can only grow in low nutrient oligotrophic seawater are therefore oversimplifications of processes that govern growth of these organisms. Some basic guidelines are given for maintenance of coral communities in aquaria.     

Pepsin fragmentation of botulinum type E neurotoxin: Isolation and characterization of 112, 48, 46, and 16 kD fragments

Controlled digestion of 150 kD single chain botulinum type E neurotoxin with pepsin atpH 6.0 produced 112, 48, 46, and 16 kD fragments. These were chromatographically purified; their locations in the 1300 amino acid residue long neurotoxin were determined by identifying the amino terminal 10 residues of 112 and 48 kD fragments, 50 residues of 46 kD fragment, and 59 residues of 16 kD fragment. The 48 and 112 kD fragments contain the N-terminal segment of the neurotoxin (i.e., residue no. 1 to 425 and 1 to 990, respectively), the 46 kD fragment corresponds to 407 residues of the C-terminal region, and the 16 kD fragment contains the 140 residues from a segment nearer to the C-terminus. The 48 kD fragment is similar to the 50 kD N-terminal light chain of the 150 kD dichain neurotoxin, which is generated by tryptic cleavage of the 150 kD single chain neurotoxin, and is separated from the 100 kD C-terminal heavy chain by dithiothreitol (DTT) reduction of an intrachain disulfide bond in the presence of 2 M urea (Sathyamoorthy and DasGupta,J. Biol. Chem. 260, 10461, 1985). The pepsin-generated 48 kD fragment, unlike the light chain, was isolated without exposure to DTT and urea. The single chain 112 kD fragment following trypsin digestion yielded 48 and 60 kD fragments that were separable after DTT reduction of the intrachain disulfide which links them. The N-terminal residues of the smaller fragment were identical to that of the single chain 150 kD neurotoxin; the single chain 112 kD fragment is therefore the neurotoxin minus the 50 kD C-terminal half of the heavy chain. The biological activities of the 48 and 112 kD fragments can be demonstrated in permeabilized PC12 cells (Lomnethet al., J. Neurochem. 57, 1413, 1991); they inhibit norepinephrine release.     

The supramolecular organization of red algal cell membranes and their participation in the biosynthesis and secretion of extracellular polysaccharides: a review

Summary The relationship between the supramolecular organization of red algal cell membranes and the biosynthesis and secretion of the cell wall skeletal and matrix polysaccharides is reviewed. Freeze-fracture studies have revealed that organized macromolecular structures — linear terminal complexes and tetrads — are present on the plasma membrane and on membranes of the endomembrane system. The linear terminal complexes seem to be involved in the biosynthesis, assembly, and orientation of the cellulose microfibrils and the tetrads in the synthesis of the matrix polysaccharides. It is shown how the research on the supramolecular organization of cell membranes has increased the knowledge on the biosynthesis and secretion of the extracellular crystalline and non-crystalline polysaccharides in red algae. In this review, the progress to date is discussed.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Longitudinal and temporal trends in the water chemistry of the North Branch of the Moose River

Surface water acidification is potentially a problem in regions with low ionic strength drainage waters. Atmospheric deposition of sulfuric acid has generally been implicated as the causative agent of this problem, although other sources of acidity may contribute. The Adirondack region of New York State is an area with acid-sensitive surface waters and an abundance of acidic lakes. The intent of this study was to evaluate the processes regulating the acid/base chemistry of a series of lakes draining a large heterogeneous watershed in the Adirondack region of New York.The study site, the North Branch of the Moose River, is heterogeneous in its soil and geological characteristics. This variability was reflected through differences in water chemistry that occurred within the basin. The northern headwaters generally drain subcatchments with shallow, acidic soils. The resulting water chemistry was acidic (equivalence of acidic anions exceeded equivalence of basic cations) with high concentrations of Al and dissolved organic carbon (DOC). As this water migrated through a large lake (Big Moose Lake) with a moderate hydrologic retention time (0.5 yr), considerable loss of DOC was evident.As acidic water was transported through the drainage area, it mixed with waters that were enriched in concentrations of basic cations from the eastern subbasins. As a result, there was a successive increase in the acid neutralizing capacity (ANC) and a decrease in Al concentrations as water migrated from the northern reaches to the outlet of the watershed.In addition to these general trends, short-term changes in water chemistry were evident. During low flow summer periods concentrations of basic cations were elevated, while concentrations of SO ₄ ^2– and NO ₃ ^– were relatively low. These conditions resulted in less acidic waters (higher ANC) with relatively low concentrations of Al. During high flow winter/spring conditions, elevated concentrations of SO ₄ ^2– and NO ₃ ^– were evident, while concentrations of basic cations were reduced resulting in low pH (low ANC) waters with high concentrations of Al.Variability in the processes regulating the pH buffering of waters was apparent through these short-term changes in water chemistry. In the northern subbasin short-term fluctuations in ANC were minimal because of the buffering of Al under low pH conditions. Seasonal changes in the ANC were more pronounced in the eastern subbasin because of the predominance of inorganic carbon buffering in the circumneutral pH waters.Lakes in the west-central Adirondacks have characteristically short hydraulic residence times and elevated nitric acid inputs. As a result these waters may be more susceptible to surface water acidification than other acid-sensitive lake districts in eastern North America. Given the apparent interregional differences, extrapolation of chemical trends in the Adirondacks to other areas may be tenuous.     

Determination of the cell adhesion specificity of Streptococcus suis with the complete set of monodeoxy analogues of globotriose

Streptococcus suis causes meningitis and other serious infections in pigs and humans, and binds to host cell globotriosylceramide. In order to determine the essential hydroxyls involved in binding, the complete set of monodeoxy derivatives of the receptor trisaccharide Gal1-Gal1-4Glc were tested as inhibitors of bacterial hemagglutination. Removal of the 4-, 6, 2 or 3-hydroxyls abolished inhibitory activity, which indicated that they were critically involved in binding. The same results were obtained using synthetic lipid-linked monodeoxy derivatives of the trisaccharides in a thin-layer overlay assay. The P_N and P_O subtypes of the S. suis adhesin showed similar binding patterns. The hydroxyls of the glucose moiety were not critical for binding, although the adhesin binds better to the trisaccharide Gal1-4Gal1-4Glc than the disaccharide Gal1-4Gal.     

Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of Human Immunodeficiency Virus-1 infectivity in vitro

Heparins/heparan sulfates modulate the function of proteins and cell membranes in numerous biological systems including normal and disease processes in humans. Heparin has been used for many years as an anticoagulant, and anticoagulant heparin-mimetics were developed several decades ago by chemical sulfation of non-mammalian polysaccharides, e.g., an antithrombotic sulfated xylan. This pharmaceutical, which comprises a mixture of sulfated oligoxylans, also mimics most other biological actions of natural heparins in vitro, including inhibition of the human immunodeficiency virus, but the molecular basis for these actions has been unclear. Here, numerous Components of the sulfated oligoxylan mixture were isolated and when bioassayed in the case of anti-HIV-1 infectivity revealed that a structural specificity underlines the capacity of sulfated xylan to inhibit HIV-1, rather than a non-specific mechanism. Components were isolated by chromatographic fractionation through Bio-Gel P10 in 0.5M ammonium bicarbonate. This fractionation revealed an elution range associated with apparent molecular weights of 22 000 to <1500 relative to standard heparin and heparan sulfates and newly prepared sulfated oligosaccharide standards. Components were characterized by metachromatic absorption spectroscopy, ultracentrifugation, GlcA analysis, and potency against HIV-1 infectivity, both in the tetrazolium cytotoxicity assay and in syncytium-forming assays, in CD4-lymphocytes. Structural specificity was indicated by the differential potencies exhibited by the Components: Highest activity (cytotoxicity) was exhibited by Components in the chromatographic region 5500 in mass (50% effective (inhibitory) concentration=0.5–0.7 g ml–1 in the first fractionation series, and 0.1–0.5 g ml–1 in a second series). The potency declined sharply below 5400 in mass, but with an exception; a second structure exhibiting relatively high potency eluted among low-mass oligosaccharides which had an average size of a nonomer. Components displayed differential potencies also against the syncytium-forming infectivity of HIV-1. The high potency against syncytium-formation was retained by Components down to a minimum size of about 4500 in mass, smaller than the 5400 required above. One in ten of the 1,4-linked xyloses in the native xylan are substituted with a monomeric 1,2 DGlcA branch. We have speculated that pharmaceutical actions of sulfated xylan might be related to structures involving the -D linked substituents and this was examined using a space-filling model of a sulfated octaxylan and by analyses of Components for GlcA content. Understanding structure/function relations in the heparin-like actions of these agents would be of general significance for the careful examination of their potential clinical usefulness in many human processes modulated by heparins, including AIDS.     

Heterogeneous binding and killing behaviour of human γ/δ-TCR+ lymphokine-activated killer cells against K562 and Daudi cells

Effector-target conjugates, formed by coincubation of lymphokine-activated killer (LAK) cells with either K562 or Daudi cells, were separated from single cells by Percoll sedimentation. The occurrence of various CD molecules (CD3, CD56, CD57, CD16, /-TCR) was compared in both fractions. Only LAK cells expressing the / T cell receptor (TCR) were found in a significantly increased percentage in fractions containing conjugates indicating that /-TCR⁺ LAK cells were preferably bound to target cells at the time of separation. In order to determine whether /-TCR⁺ LAK cells also show a preferred killing activity against the targets, cultures enriched with or depleted of /-TCR⁺ cells were established. Against K562 cells, /-TCR⁺-enriched cultures showed a greatly reduced killing activity compared to LAK bulk cultures or cultures depleted of /-TCR⁺ cells. Using Daudi cells as targets the enriched fraction revealed a slightly increased killing activity compared to bulk cultures or depleted fractions. Preincubation of /-TCR⁺ LAK cells with anti-/ or anti-CD3 mAb resulted in a distinct increase of the killing activity against K562 cells, but in only a slightly enhanced activity against Daudi cells. It is postulated that /-TCR⁺ LAK cells use the same adhesion mechanism for both targets but that only Daudi cells express a specific ligand for the /-TCR. Occupation of the /-TCR/CD3 complex by mAb, however, seems to substitute for the absent epitope on K562 cells by eliciting stimulatory signals in /-TCR⁺ LAK cells which, in combination with the binding stimulus, trigger cytolytic activity.This work was supported by the Hartmann-Müller Foundation, Zürich     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

Synthesis of nanophase hydroxyapatite by a <Emphasis Type="Italic">Serratia</Emphasis> sp. from waste-water containing inorganic phosphate

Synthesis of nanophase hydroxyapatite (HA) on a bacterial surface was achieved at the expense of CaCl₂ and inorganic phosphate (Pi). After initial nucleation, calcium was precipitated on and around the cells as calcium phosphate at the expense of inorganic phosphate in the challenge solution, with no precipitation in cell-free controls. HA was also biomanufactured using inorganic phosphate ions scavenged from a phosphate-containing waste-water. With additional Ca²⁺, the concentration of phosphate was decreased from 0.27 (25ppm) to 0.02m (2ppm) in the waste-water. Crystals of calcium phosphate manufactured by the cells were located by scanning electron microscopy (SEM) and identified as HA by X-ray powder diffraction, with an average crystal size calculated as 25nm. Possible application of bioHA as a biomaterial and implications for one-step `waste-into product' are discussed.     

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.     

Myosatellite cells associated with different muscle fibre types in the Atlantic hagfish (Myxine glutinosa,L.)

Summary The incidence of myosatellite cells associated with white and red muscle fibres of the parietal muscle and red fibres of the craniovelar muscle was estimated by quantitative electron microscopy in the Atlantic hagfish (Myxine glutinosa, L.). Myosatellite cell nuclei constitute 3, 11 and 23 % of the total number of nuclei inside the basal lamina of the three types of muscle fibres, respectively. However, the total number of nuclei is highest in white fibres, most of the nuclei belonging to striated muscle cells. Myosatellite cell profiles in transverse sections constitute 23, 41 and 61 % of the number of muscle fibre profiles of the three types, respectively. The intervals between adjacent myosatellite cells are 135 m in white fibres, 55 m in red parietal fibres, and only 25 m in craniovelar fibres. Since craniovelar fibres are also comparatively thin, myosatellite cells constitute a significant fraction of the volume inside the basal lamina in these fibres. The myosatellite cells are 30–50 m long and up to 5 m thick. Some myosatellite cells possess few organelles, whereas others appear to contain many free ribosomes, granular endoplasmic reticulum, prominent Golgi apparatus and lysosome-like bodies.This investigation was supported by the Norwegian Research Council for Science and the Humanities (NAVF grant No. C20.30–37). The authors are indebted to Jorunn Line Vaaland and Berit Branil for technical assistance, and to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supplying the hagfish     

Alzheimer's Aβ1–40 Peptide Modulates Lipid Synthesis in Neuronal Cultures and Intact Rat Fetal Brain Under Normoxic and Oxidative Stress Conditions

The effect of amyloid (A), the major constituent of the Alzheimer's (AD) brain on lipid metabolism was investigated in cultured nerve cells and in a fetal rat brain model. Differentiated (NGF) and undifferentiated PC12 cells or primary cerebral cell cultures were incubated with [¹⁴C]acetate in the absence or presence of A_1–40. Incorporation of label into lipid species was determined after lipid extraction and TLC separation. Phosphatidylcholine (PC) and phosphatidylserine (PS) synthesis was increased by A_1–40, in a dose dependent manner, an effect which was more pronounced in differentiated PC12 cells. A significant proportion of radioactivity (5–6%) was released into the medium with a radioactivity distribution similar to that of the cellular lipids. Cholesterol and PC were the highest labeled medium lipids. Increasing A_1–40 concentration up to 0.1 g/ml in cerebral cells but not in PC12 cells, caused a relative increase (1.5 fold) in release of PS, while that of PE decreased. Stimulation of PS release may possibly be associated with apoptotic cell death. A_1–40 peptide (5 g) was administered intraperitonealy into rat fetuses (18 days gestation) along with [¹⁴C]acetate (2Ci/fetus). After 24 h, the maternal-fetal blood supply was occluded for 20 min (ischemia) followed by 15 min reperfusion. Fetuses were killed and liver and brain tissue subjected to lipid extraction and radioactivity determination after TLC. A_1–40 peptide increased synthesis of different classes of lipids up to 20–40% in brain tissue compared to controls. Labeling of liver lipids was decreased by A_1–40 by 20–30%. A general decrease in synthesis of lipids was observed after ischemia/reperfusion. Our data suggest that A_1–40 peptide regulates normal lipid biosynthesis but under ischemia it compromises it. The latter finding may confirm the oxidative stress etiology in AD and suggests that A_1–40 modulation of lipid metabolism may have Alzheimer's pathological relevance, particularly at high peptide concentrations.     

Calcium control of differentiation in Phytophthora palmivora

A method has been developed for the preparation of zoospores from Phytophthora palmivora which allows the ionic composition of the suspension medium to be closely controlled. Sub-micromolar concentrations of calcium ions have been shown to play a key role in maintaining the zoospore state and in the transition to the cyst stage. Restriction of free Ca²⁺ to between 0.2 and 1 M resulted in zoospores which could be maintained for several hours before they finally encysted and germinated. When exposed to citrus-pectin, or 3 mM SrCl₂, or to vigorous shaking, these zoospores underwent rapid synchronous encystment. At free Ca²⁺ concentrations below 0.1 M, zoospores lysed slowly. If exposed to inducers of encystment before lysis had occurred, the zoospores failed to respond to pectin or to vigorous shaking. However, they did differentiate in response to SrCl₂ addition. Provided the free Ca²⁺ was maintained between 0.02 and 0.2 M, zoospores survived gentle centrifugation, a procedure which previously had resulted in encystment.Abbreviations IM (ion-mix) release medium containing 100 M KCl, 10 M CaCl₂, and 10 M MgCl₂     

Evidence for expression of a single distinct form of mammalian cysteine dioxygenase

Summary. Cysteine dioxygenase (CDO) plays a critical role in the regulation of cellular cysteine concentration. Because multiple forms of CDO (23kDa, 25kDa, and 68kDa) have been claimed based upon separation and detection using SDS-PAGE/western blotting (with antibodies demonstrated to immunoprecipitate CDO), we further investigated the possibility of more than one CDO isoform. Using either rabbit antibody raised against purified rat liver CDO or against purified recombinant his₆-tagged CDO (r-his₆-CDO) and using 15% (wt/vol) polyacrylamide for the SDS-PAGE, we consistently detected the 25kDa band, but never detected a 68kDa band, in rat liver, kidney, lung and brain. Nondenatured gel electrophoresis of r-his₆-CDO yielded a molecular mass estimate of 25.7kDa and no evidence of dimerization. Mass spectrometry of r-his₆-CDO yielded two peaks with molecular masses of 24.1kDa and 24.3kDa. Anion-exchange FPLC of r-his₆-CDO also gave two peaks, with the first containing CDO that was 7.5-times as active as the more anionic form that eluted second. When the two peaks recovered from FPLC were run on SDS/PAGE, the first (more active) CDO fraction yielded two bands (perhaps as an artifact of SDS/PAGE), whereas the second (less active) CDO fraction yielded only the 23kDa band. We conclude that the physiologically active form of CDO is the 25kDa (i.e., 23.5kDa based on mass spectrometry) monomer and that this active form is probably derived by post-translational modification of the 23kDa gene product.     

The Effect of Salicylic Acid on Peroxidase Activity in Wheat Calli Cocultured with the Bunt Pathogen Tilletia caries

The effect of salicylic acid (SA) on peroxidase activity in wheat (Triticum aestivum L.) calli cocultured with the bunt pathogen Tilletia caries was studied. Fungal infection was shown to activate cytoplasmic peroxidase. SA suppressed total peroxidase activity but did not inhibit the peroxidase with pI 9.8. A novel chitin-specific peroxidase with pI 3.5 appeared after the SA treatment. The infection of SA-treated cells with Tilletia caries activated the isoenzymes with pI 3.5, 4.8, and 7.5 and stimulated their secretion into the culture medium. The ability of SA to control wheat peroxidase activity during pathogenesis is discussed. The important role of this control in plant defense responses to the bunt pathogen is emphasized.