Cell Surface Sialoglycoproteins of Cultured Rat Cerebellar Interneurons

Abstract: The sialoglycoproteins of cultures of relatively pure rat cerebellar interneurons were labelled by NaIO₄ oxidation/NaB ³H₄ reduction. The labelled molecules were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate followed by fluorography. Faint labelling could be detected in three components if cells were labelled without any oxidation. In young cultures, oxidation by galactose oxidase alone failed to reveal any additional bands. After oxidation by NaIO₄ or galactose oxidase in the presence of neuraminidase, many more components were labelled. After NaIO₄ oxidation, about 80% of the cell-associated radioactivity could be removed by treating the cells with neuraminidase, which left the cells more than 95% viable. The majority of the bands seen after neuraminidase treatment were substantially reduced when compared with untreated controls, supporting a surface localisation of these molecules. Reproducible developmental changes were seen in the profiles of bands labelled by NaIO₄/NaB ³H₄ in time course studies of cultures up to 8 days in vitro . Some bands became more prominent, and others disappeared. The gel profiles of the neuron cultures were quite distinct from those of cerebellar astrocyte cultures, which contain all the cell types likely to be contaminants of the neuron cultures.     

Genetic polymorphism of the vitamin D binding protein (Gc) in the musk shrew (Suncus murinus)

In the musk shrew ( Suncus murinus ), the electrophoretic bands in the post-albumin region were identified as vitamin D binding protein (Gc) by the [³HI vitamin D₃ binding method. Three Gc phenotypes were distinguished from each other: a single faster band (Gc-A), a single slower band (Gc-B) and the double bands (Gc-AB). Results of mating experiments indicated that the Gc-A and Gc-B are controlled by two codominant alleles, Cc ^a and Gc ^b at an autosomal locus ( Cc ), respectively. It was noticed that, in the Gc-AB phenotypes, the Gc-B band was constantly more intense than the Gc-A band in the protein staining. The same tendency was also observed btween the homozygous Gc-A and Gc-B bands, and further, radioactivity of the Gc-B bound with [³H] vitamin D₃ was about twofold higher than that of the Gc-A. These results suggest that the Gc^b yields its protein product twofold more than the Gc ^a. No cross-reaction between the shrew proteins and a rabbit anti-human Gc protein was observed.     

Gas exchange and metabolite fluctuations in green and yellow bands of variegated leaves of the monocotyledonous CAM species Agave americana

The variegated leaves of the Crassulacean acid metabolism (CAM) species Agave americana have a large central longitudinal green band with narrow yellow bands on either side. The yellow bands had 97% less pigment content, 84% lower ribulose‐1,5‐bisphosphate carboxylase/oxygenase activity, but only 20% lower phosphoenolpyruvate carboxylase activity than the green band. The green bands exhibited gas exchange typical of CAM plants, with most CO₂ uptake occurring at night, leading to a daily net CO₂ uptake of 127 mmol m⁻² day⁻¹. The yellow bands had some nighttime net CO₂ uptake but a larger loss during the daytime, indicating that they were sink tissues. Nocturnal citrate and malate accumulations for the yellow bands were 65 and 75%, respectively, of those of the green bands; sucrose supported 64‐83% of their nocturnal acid accumulation. This is the first evidence that agaves, which are malic‐enzyme‐type CAM plants, use sucrose as the carbon source for nocturnal acid accumulation. About 44% of the carbon demand of the yellow bands can be supplied by sucrose diffusing via the symplast from the adjacent green band, about 25% from fructose and glucose diffusion, and some via the apoplast.     

Analysis of Hardening of the Egg Envelope (Chorion) of the Fish, Oryzias latipes

Hardening of the chorion of medaka eggs was quantitated in terms of the solubility of its constituent proteins. After activation of unfertilized eggs with the Ca²⁺-ionophore A23187, hardening of chorion (named ionophore-activation hardening) proceeded and 60 min after activation the solubility of the proteins in 1 N NaOH had decreased to 20% of that of proteins of unhardened chorions. On SDS-PAGE, the chorions of unfertilized eggs gave four major protein bands (150, 83, 78 and 51 K). After Ca²⁺-ionophore activation, new two protein bands (135 and 61 K) appeared, with concurrent disappearance of all the original bands except the 51 K band. Isolated chorions of unfertilized eggs were also hardened by Ca²⁺and 60 min after addition of Ca²⁺the solubility of their proteins in 1 N NaOH had decreased to about 45% of that originally. During this type of hardening (named 'Ca²⁺-hardening), however, the SDS-PAGE pattern of the proteins remained unchanged. Therefore, there are two mechanisms of hardening. The 'ionophore-activation hardening was inhibited by cadaverine. When chorions were isolated 20 min after Ca²⁺-ionophore activation and kept in Ca²⁺-free conditions, the 'ionophore-activation hardening process was arrested: further hardening was resumed on addition of Ca²⁺to the medium. These results suggest the presence of some hardening machinery in isolated chorions.     

The Phosphorylation of Proteins in Hippocampal Slices: Effects of Noradrenaline and of Pretreatment with Kainic Acid

Abstract: Hippocampal slices were incubated in the presence of [³²P]P_i, and protein phosphorylation was examined by means of sodium dodecyl sulfate-gel electrophoresis. Incubation for at least 30 min with 300 μCi of [³²P)P_i/brain slice gave rise to the phosphorylation of 8–10 protein bands. Most of these bands showed enhanced phosphorylation in response to noradrenaline. The basal phosphorylation of kainic acid-pretreated hippocampal slices was enhanced two- to threefold compared with controls. There was also an additional increase in kainic acid-pretreated slices in the response to noradrenaline. 8-Br-Cyclic AMP and phosphodiesterase inhibitors, such as papaverine or isobutylmethyl-xanthine, had no effect on the phosphorylation patterns.     

Induction of proteins during phage reactivation induced by UV irradiation, oxygen and peroxide in Bacteroides fragilis

Abstract Phage reactivation systems in Bacteroides fragilis were induced by far-UV irradiation, O₂ and H₂O₂. These three treatments also induced the synthesis of 3, 6, and 4 protein bands, respectively, which were easily detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Two proteins with apparent M _r s of approx. 90 000 and 70 000 were induced by all three treatments. Caffeine completely inhibited UV- and O₂-induced phage reactivation and prevented the synthesis of the M _r 90 000 and M _r 70 000 proteins. The results suggest that these two proteins may be involved in phage reactivation processes induced by UV, O₂ and H₂O₂ in B. fragilis .     

Aerotaxis in Desulfovibrio

Aerotaxis of two sulphate-reducing bacteria, the freshwater strain Desulfovibrio desulfuricans CSN (DSM 9104) and the marine strain Desulfovibrio oxyclinae N13 (DSM 11498), was studied using capillary microslides, microscopy and oxygen microsensors. The bacteria formed ring-shaped bands in oxygen diffusion gradients surrounding O₂ bubbles, which were placed into anoxic sulphate-free cell suspensions in capillary microslides. The radial expansion of the oxic volume by diffusion was stopped by aerobic respiration. Bands were formed by cells avoiding high O₂ levels near the O₂ bubble, as well as by cells entering from the surrounding anoxic zone. At the inner edge of the bands, O₂ levels of up to 20% air saturation (50 μM O₂) were found, while the outer edge always coincided with the oxic–anoxic interface. Ring diameters and O₂ concentrations at the inner edge of the band depended on the cell density and the strain used in the suspension. Band formation did not occur in the absence of an electron donor (5 mM lactate) or when N₂ gas bubbles were used. Both strains were highly motile with velocities of ≈ 32 μm s⁻¹ during forward runs, and 7 μm s⁻¹ during backward runs respectively. Within the bands, cells moved in circles of about 20 μm diameter, while cells outside the band exhibited straighter or only slightly bent traces. It is concluded that the capacity of respiration at high rates and the positive and negative aerotactical responses of Desulfovibrio provide an efficient strategy for removing O₂ from the habitat in situations where sufficient electron donors and high cell densities are present.     

Two forms of phycobilisomes in the Antarctic red macroalga Palmaria decipiens (Palmariales, Florideophyceae)

The phycobilisomes (PBS), the light-harvesting antennae, from the endemic Antarctic red macroalga Palmaria decipiens were isolated on discontinuous sucrose gradients in two discrete bands and not in one as expected. To exclude methodical faults, we also isolated PBS from the temperate Palmaria palmata and the unicellular red algae Porphyridium cruentum and Rhodella violacea . In P. palmata the PBS were separated in two discrete bands, whereas the PBS from Porphyridium and Rhodella were found in one band. The double-banded PBS (PBS_up and PBS_low) from P. decipiens were further characterized by absorption and fluorescence spectroscopy, native and SDS-PAGE as well as by negative staining. The phycobiliproteins RIII-phycoerythrin, RI-phycocyanin and allophycocyanin were identified and 3 γ -subunits were described. The PBS_up and PBS_low showed no significant differences in their absorption spectra and phycobiliprotein ratios although the negative stained PBS_low were smaller. Differences were found in their low molecular mass subunit complexes, which are assumed to be r-phycoerythrin. The polypeptide pattern of the PBS_up and PBS_low showed no differences in the molecular masses of their subunits and linker polypeptides, but in their percentage distribution. The results suggest that the PBS_low is a closer packed and PBS_up a little more loosely aggregated hemiellipsiodal PBS form. We discuss the ecophysiological function of two PBS forms in P. decipiens and suggest advantages in the rapid acclimation to changes in environmental light conditions.     

RAT BRAIN TUBULIN: SUBUNIT HETEROGENEITY AND PHOSPHORYLATION

Abstract— Evidence for multiple forms of the α and β subunits of tubulin isolated from rat brain has been obtained by means of SDS polyacrylamide gel electrophoresis, isoelectric focusing, and SDS hydroxylapatite column chromatography. Fourteen distinct bands, localized near pH 5.4, were formed when tubulin was subjected to isoelectric focusing in a gradient established with a very narrow range ampholyte mixture. Three tubulin subunits, a₁., α₂, and β, were separated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in a second dimension. The β subunit was more acidic than the α subunits. Brain sections were incubated in tissue culture medium containing ³²P₁ and radiolabeled tubulin was subsequently isolated and subjected to electrophoresis. Only the β subunit was labeled. All radioactivity was associated with two or three adjacent bands on isoelectric focusing gels.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Proteins that interact with GTP in Streptomyces griseus and its possible implication in morphogenesis

Abstract By cross-linking with [α-³²P]GTP or [γ-³²P]GTP with or without UV treatment, several proteins of Streptomyces griseus were shown to interact with GTP in specific ways. After gel electrophoresis, 19 bands of radioactivity were found; 12 bands were assigned as GTP-binding proteins and 6 bands as phosphorylated proteins. One band was assumed to be a guanylylated protein. The profile of radioactive bands was similar between cells prepared from liquid or solid culture, but markedly different between growth phases. A mutant (strain M-1) defective in aerial mycelium formation, which was originally found as a decoyinine-resistant isolate, was found to have a different profile of phosphorylated proteins.     

Analysis of lipopolysaccharides of Bacteroides fragilis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroblot transfer

Abstract Lipopolysaccharides (LPS) from three strains of Bacteroides fragilis were run on SDS-polyacrylamide gels and stained with silver. Each LPS produced a similar pattern, consisting of a series of regularly spaced discrete bands which decreased in intensity as they increased in M _r value. Electroblot transfer from duplicate SDS gels onto nitrocellulose membrane were reacted with antisera raised to whole cells of two of the strains and antigens were visualised with horse-radish peroxidase-antirabbit-IgG conjugate and colour reagent. Results revealed that the two lowest M _r bands of the LPS preparation (rough LPS) represented common antigens.     

Population ecology of the white-nosed coati (Nasua narica) on Barro Colorado Island, Panama

The white-nosed coati, Nasua narica , is a common Neotropical carnivore with a social structure of band-living adult females and solitary adult males. A coati population on Barro Colorado Island, Panama, was studied over a four-year period by mark-recapture, radiotelemetry. and direct observation of habituated individuals. The population density was approximately 51.5 individuals/km² and the sex ratio was 1:1. Band size varied from six to 26 individuals (mean = 15.3) with extensive fluctuation within and between years. Mean foraging group size was smaller (7.2 individuals) than population group size, and fluctuated with food availability, synchronous parturition, and the emigration of mature males. Mean home-range size of six bands was 0.33 km², and ranges of adjacent bands overlapped from 0–66%. One band fissioned during the study; however, the resulting bands did not disperse from the original home range. Seven adult males had a mean home-range size of 0.37 km², each extensively overlapping the home ranges of several other males. Observations of 10 adult males whose natal bands were known indicate that when males disperse they do not simultaneously leave the band's home range. Rather, their home ranges remain within or broadly overlapping those of their natal bands. This dispersal pattern is unusual within the order Carnivora.     

Differential Accumulation of Translatable mRNA Species During a Specific Host-pathogen Interaction Between Puccinia recondita tritici and Triticum aestivum

The time-course of mRNA induction in specific cultivar-race combinations was examined using a criss-cross interaction system consisting of nearly isogenic lines of wheat (Sinvalocho M.A. and Gamma 1R) and genetically related pathogenic races of leaf rust Puccinia recondita f. sp. tritici . Infection stimulated the differential accumulation of mRNA species, identified by in vitro translation, after only three days of inoculation with rust spores. Comparisons of different host-pathogen combinations showed polypeptide pattern changes likely to be associated with gene-for-gene relationships. At least two specific mRNAs which code for polypeptide bands of 34 and 24 kDa are associated with the compatible interaction mediated by genes A₁,/A₂ from Gamma 1R wheat line and virulence gene p_t/p₂ of rust race FO₁. Comparisons made using a mutant clone of rust, which elicits an inverse criss-cross relationship with the same wheat lines, are consistent with the proposed specificity of the detected changes.
In addition, two wheat mRNAs (coding for polypeptide bands of 20.5 and 32.5 kDa) were elicited by infection with rust race FO₁ regardless the plant genotype or reaction type. A cDNA clone involved in this kind of race specific induction has been isolated.     

Survival strategies of plasmid-carrier and plasmidless Escherichia coli strains under illuminated and non-illuminated conditions, in a fresh water ecosystem

A Comparative study, in illuminated and non-illuminated systems, was made to determine the survival strategies of plasmid-carrier and plasmidless bacteria in sterile river water. Two strains of Escherichia coli from river water were selected: one plasmidless, EC1, and one antibiotic-resistant strain, EC7, which showed plasmid bands. By matings with EC7 as donor and E. coli K12 strain J62 as recipient, transconjugants were generated, the J62₇ strain, which showed both antibiotic resistance and plasmid bands. Ethidium bromide curing of the EC7 strain generated the EC7₂ strain which showed a partial loss of resistance and a reorganization of plasmid bands. Under non-illuminated conditions the total number of cells detected by direct count and the number of culturable cells (injured and non-injured cells) remained practically constant throughout the period of incubation. In the illuminated systems, however, the number of cfu decreased in four of the five strains studied. The greatest decreases are those of the J62 strain, followed by those of the J62₇, EC1, EC7₂ and EC7 strains. Differences in survival strategies as a consequence of the presence or absence of plasmids are discussed.     

Evidence for Chromosome Endoreduplication in Eudorina californica, a Colonial Alga

Several nuclear events seen during the cleavage period in Eudorina suggest that chromosome endoreduplication, proportional to the number of cells to be produced, may occur in the gonidia prior to cleavage. Presumably the DNA concentration is reduced to the haploid level during rapid, successive divisions of the cleavage period.
To test this hypothesis, I determined DNA content of gonidia as they grew from 4 μm to 38 μm in diameter between cleavage periods. During growth from 4 μm to 8 μm in diameter, the DNA concentration remained at the haploid level of 0.17 pg/cell. As gonidia in 64 cell colonies continued to grow from 8 μm to 33 μm in diameter, their DNA concentrations increased 60-fold.
Analysis of the Eudorina DNA by equilibrium centrifugation in CsCl showed only 2 bands with buoyant densities of 1.721 g/cm³ and 1.699 g/cm³, presumed to be nuclear and chloroplast, respectively, on the basis of labelling with ³H-thymidine and ³H-adenine. The 8: 2 ratio of the two bands did not change with increase in cell size and no other bands were detected, suggesting that both nuclear and chloroplast DNAs were synthesised proportionately prior to the cleavage period.     

Genetic markers linked to quantitative traits in poultry

This study utilized DNA fingerprints and crosses of two genetically distinct lines of layer-type chickens to identify genetic markers linked to quantitative trait loci (QTL). In phase I, back-cross (BC¹) hens were separately ranked for each of eight traits and then blood pools were produced in groups along each phenotypic distribution. The DNA was isolated from the blood pools and used in a gradient analysis to screen for DNA fingerprint bands that exhibited intensity gradients associated with the phenotypic traits. To identify linkage of bands with QTL and to estimate band effects, F₂ progeny were produced in phase II from the phase I BC, population. A single-trait animal model was used for analysis of associations of all individual DNA fingerprint bands of sires and their progeny phenotypic performance. Twenty fingerprint bands, only two of which had shown trait-associated gradients in phase I, were identified by the animal model analysis of the progeny test as QTL linked (P≤005) to specific traits of growth, reproduction and egg quality. These 20 bands warrant further study as potentially valuable molecular markers for QTL.     

A 30-kDa Protein in the Surface Complex and Flagella of Euglena has Protein Kinase Activity

ABSTRACT A protein kinase (PK) was partially purified from NaCl extracts of the cell surface complex of Euglena using DEAE-cellulose chromatography. Tubulins extracted either from flagella or from the cell surface complexes of Euglena were readily phosphorylated when incubated with [γ-³²P]-ATP and the PK. Protein kinase activity was augmented with 5 mM Mn²⁺ or Mg² and was inhibited or had greatly reduced activity with 5 mM Ca²⁺, Co^2-, Cu²⁺ or Zn²⁺. Incorporation was much lower when [γ-³²P]-GTP was the phosphate donor. Serine and threonine were the major radiolabeled phosphoamino acids in tubulins; label was also found in phosphotyrosine. Alpha-tubulin solubilized from flagella was a relatively poor substrate for the PK, but a Euglena α-tubulin cDNA overexpressed as a Trx-fusion protein incorporated [γ-³²P]-ATP into serine and threonine when incubated with cell surface extracts. Alpha- and β-tubulins from cell surface complexes were equally good substrates for the PK. No incorporation was observed in intact microtubules either from the cell surface complex or from isolated flagella. In-gel assays identified a polypeptide of about 30 kDa that phosphorylated tubulins in extracts of both flagella and the cell surface complexes, and dephosphorylated casein was a competitive substrate for the partially purified kinase. In vivo incubation with [³²P]-orthophosphate produced numerous radiolabeled bands in acrylamide gels of NaCl extracts of the cell surface complex, but none of these bands could be positively related to tubulins extracted from surface complex microtubules.     

Major histocompatibility complex (MHC) related cDNA probes associated with antibody response in meat-type chickens

The major histocompatibility complex (MHC) region was examined as a set of candidate genes for association between DNA markers and antibody response. Intercross F₂ families of chickens were generated from a cross between high (HC) and low (LC) Escherichia coli _i antibody lines. Restriction fragment length polymorphism (RFLP) analysis was conducted by using three MHC-related cDNA probes: chicken MHC class IV ( B-G ), chicken MHC class I ( B-F ), and human MHC-linked Tap2 . Association between RFLP bands and three antibody response traits ( E. coli , sheep red blood cells and Newcastle disease virus) were determined by two methods: by statistically analyzing each band separately and also by analyzing all bands obtained from the three probes by using multiple regression analysis to account for the multiple comparisons. The MHC class IV probe was the highest in polymorphisms but had the lowest number of bands associated with antibody response. The MHC class I probe yielded 15 polymorphic bands of which four exhibited association with antibody response traits. The Tap2 probe yielded 20 different RFLP bands of which five were associated with antibody production. Some Tap2 bands were associated with multiple antibody response traits. The multiband analysis of the three probes' bands revealed more significant effects than the analysis of each band separately. This study illustrates the efficacy of using multiple MHC region probes as candidate markers for quantitative trait loci (QTLs) controlling antibody response in chickens.     

Growth of methanotrophs in methane and oxygen counter gradients

Abstract A gel-stabilized system with counter gradients of CH₄ and O₂ was used to grow methanotrophs from wetland, agricultural and forest soils and lake sediment. Columns of semi-solid nitrate- or ammonium-minerai salts medium were continuously flushed at opposite ends with CH₄ and O₂ to create opposing concentration gradients of the two gases. Methanotrophs grew from all samples except forest soil, and were visible as thin bands after 5 to 15 days of incubation. The position of growth was CH₄ and O₂ concentration-dependent and occurred at the point of maximum possible CH₄ oxidation, where both substrates were completely consumed. Evidence was obtained for denitrification and nitrification activities concomitant with CH₄ oxidation. This approach may be useful to isolate methanotrophs with different CH₄ and O₂ requirements and to study their interactions with other groups of bacteria in nature.