In vitro and in vivo regulation of chloroplast glyceraldehydes-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. I. Purification and properties of isozymes

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13) was purified from leaves of Chenopodium rubrum L. Aggregated (≥ 10⁶) and disaggregated (165 × 10³) molecular weight forms were obtained by gel filtration in the presence of NAD⁺ and NADP⁺, respectively. The disaggregated enzyme was separated into two isozymes by inverse ammonium sulphate gradient solubilization: "NADP-GPD I" was homotetrameric (subunit molecular weight 39 × 10³); "NADP-GPD II" was heterotetrameric (subunit molecular weights 39 × 10³ and 43 × 10³). Isoelectric focusing of the isozymes, both aggregated and disaggregated, revealed two isoelectric forms in each case, at 4.3 and 7.7. Chloroplast GPD was "NADP-suppressed" in crude extracts due to partial oxidation, incubation with dithioerythritol restored full activity.     

CELL SURFACE PROTEINS OF CULTURED BRAIN CELLS AND THEIR RECOGNITION BY ANTI-CEREBELLUM (ANTI-NS-4) ANTISERUM

Abstract— Surface proteins of cultured young postnatal mouse cerebella and embryonic mouse cerebral hemispheres were identified by Iactoperoxidase-catalysed radioiodination and by their interaction with an anti-mouse cerebellum antiserum (anti-NS-4 serum) which recognizes surface components on brain cells. Several (8 10) iodinated polypeptides are recognized by radioautography after polyacrylamide gel electrophoresis. Their surface location was confirmed by their sensitivity to mild trypsin treatment on intact cells. Iodinated polypeptides from cells of non-nervous tissues showed a different gel pattern. Immuno-precipitates of solubilizcd surface-iodinated cerebellar cells with anti-NS-4 serum contained two prominent labeled proteins with apparent molecular weights of 200 × 10³ and 145 × 10³. These proteins were also biosynthetically labeled with [³H]leucine. The 145 × 10³ molecular weight component was also found in immunoprecipitates prepared from embryonic cerebral cells, but the 200 × 10³ molecular weight component was replaced by a broad peak with an apparent molecular weight of around 250 × 10³.     

Experimental Infection of Nosema sp. Ghose in an Unnatural Host Lesioderma sericorne

ABSTRACT. Various doses of a microsporan parasite, Nosema sp., were fed to third and fourth instar larvae of Lesioderma sericorne that infested different types of stored grains. A spore dose of 3 × 10³ spores/individual resulted in a 39% infection rate, reduction in larval and adult weights, and mean spore concentrations of 1.28 ± 0.2 × 10⁸ spores/larva and 1.1 ± 0.2 × 10⁸ spores/adult. At the above dose, mortality was not well marked (about 35% in larvae and 25% in adults). At 3 × 10⁴ spores/individual, the rate of mortality increases to 80% in larvae and 60% in adults. However, more of the pest population (88% of larvae and 73% of adults) died at a dose of 3 × 10⁵ spores/individual. This dose produced mean spore concentrations of 3.91 ± 0.2 × 10⁸ spores/larva and 2.89 ± 0.2 × 10⁸ spores/adult. Insect death was caused by heavy damage to gut epithelia and fat bodies.     

5S RNA AND TRANSFER RNA SYNTHESIS IN GERMINAL VESICLE OOCYTES AND DURING HORMONALLY-INDUCED MEIOTIC MATURATION OF STARFISH OOCYTES

The incorporation of ³H-guanosine as ³H-GMP into 5S RNA and into transfer RNA (tRNA) was examined in isolated large germinal vesicle oocytes, in isolated mature ootids and during and subsequent to hormonally (l-methyladenine)-induced meiotic maturation in the starfish, Asterias forbesii .Purified soluble RNA ¹ preparations at each stage were fractionated by electrophoresis on 10% polyacrylamide gels, while high molecular weight RNAs were resolved by subjecting total RNA samples to electrophoresis on 2.4% acrylamide+0.5% agarose gels. The results showed that large germinal vesicle oocytes, containing a single compact nucleolus, synthesize 5S RNA and tRNA as well as the previously-reported (1, 23-26) nucleolar rRNAs. In contrast, during and subsequent to hormonally-induced meiotic maturation, after germinal vesicle braekdown and nucleolar dissolution, the synthesis of 5S RNA and tRNA continues in the absence of detectable high molecular weight rRNA synthesis.     

DIRECT DETECTION OF ESCHERICHIA COLI 0157:H7 IN UNPASTEURIZED APPLE JUICE WITH AN EVANESCENT WAVE BIOSENSOR

An evanescent wave biosensor was used to detect Escherichia coli O157:H7 in unpasteurized apple juice. Light is launched from a 635 nm laser diode into silica or polystyrene optical waveguides, generating an evanescent field which extends from the waveguide surface. Fluorescent molecules within the evanescent field are excited resulting in an emission signal that the biosensor then detects and quantifies. A sandwich immunoassay was performed on the waveguides using cyanine 5 dye-labeled anti-E. coli O157:H7 antibodies for generation of the specific fluorescent signal. The lower limit of detection was between 6.0 × 10² and 6.0 × 10⁴ CFU/mL with silica waveguides and between 3.2 × 10⁴ and 3.2 × 10⁴ CFU/mL using polystyrene waveguides. One-hundred percent correct identification of true positive samples occurred at 6.0 × 10⁴ and 3.2 × 10⁴ CFU/mL for silica and polystyrene waveguides, respectively. Signals from a variety of non-E. coli O157 bacteria, including closely related enterotoxigenic strains of E. coli at concentrations of ˜ 10⁶ CFU/mL, were below the limits of detection. Assays were conducted in near real-time with results obtained within 15 min of sample processing.     

BIOSYNTHESIS OF THYROTROPIN RELEASING FACTOR BY NEWT (TRITURUS VIRIDESCENS) BRAIN IN VITRO. ISOLATION AND CHARACTERIZATION OF THYROTROPIN RELEASING FACTOR

Abstract— The in vitro biosynthesis of thyrotropin releasing factor (TRF) by hypothalamic and forebrain fragments from adult newts ( Triturus viridescens ) was studied by incubating the fragments in the presence of the radioactive amino acid precursors of thyrotropin releasing factor (TRF). The synthesized product was identified by successive chromatography and electrophoresis. A purification scheme was developed which can be applied to the processing of multiple samples labelled with l -[³H]proline and yields a TRF peak which, when derivatized with 1-fluoro-2,4-dinitrobenzene and applied to electrophoresis in acid buffer, yields a single radioactive peak corresponding to the migration of the N^im-Dnp derivative made from standard synthetic [³H]TRF. Both forebrain and hypothalamic fragments of the adult newt are capable of synthesizing a product which migrates with standard TRF after application of this purification scheme. Hypothalamic and forebrain fragments were also assayed for biological activity of TRF and it was determined that one newt hypothalamus contains approximately 200 pg TRF and that an equal amount of TRF is present in a forebrain piece of comparable size. It is concluded that newt TRF is identical to mammalian TRF (pGlu-His-Pro-NH₂) and that the storage and synthesis of this substance is not confined to the hypothalamus in this species.     

Synaptosomal Calcium Uptake Systems: Prostaglandins Are Probably Not Involved in the Regulation of Calcium Fluxes Into and Within the Nerve Endings

Abstract: ⁴⁵Ca²⁺ uptake measurements were performed on intact and osmotically lysed synaptosomes from rat brain to study the possible influence of prostaglandins (PGs) on Ca²⁺ movements into and within the nerve endings. The K⁺-induced ⁴⁵Ca²⁺ uptake of intact synaptosomes was not influenced by several inhibitors of PG synthesis. ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations was promoted by ATP and seemed to be largely attributable to mitochondria, as it was inhibited by mitochondrial poisons. This Ca²⁺ uptake was strongly reduced by PG synthesis inhibitors but also by PG precursor fatty acids. Both PG synthesis inhibitors and precursors, according to their relative efficacy in blocking Ca²⁺ uptake, were able to induce Ca²⁺ efflux from preloaded intrasynaptosomal organelles. The PGs E₂, F_2α, D₂, and thromboxane B₂ were without effect on ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations. On the basis of our results it does not seem likely that PGs influence Ca²⁺ availability by modulating Ca²⁺ fluxes into or within the nerve endings. The observed inhibitory effects of PG synthesis inhibitors and precursors on the intrasynaptosomal Ca²⁺ uptake might be due to unspecific impairment of mitochondrial functions.     

Non-Coordinated Synthesis of RNA's in Pre-Gastrular Embryos of Xenopus Laevis

The rates of syntheses of 18S and 28S rRNA, 5S RNA, capped mRNA and 4S RNA were determined in isolated cells from pre- and post-gastrular embryos of Xenopus laevis. The rate of rRNA synthesis per nucleolated cell Mas about 0.2 pg/hr, or about 5.5 × 10⁴ molecules/hr at the blastula stage, and this value remained constant in later stages. At the blastula stage, about 30 molecules of 5s RNA, 10 molecules of capped mRNA and 900 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA. These values were all greatly reduced during the gastrula stage, and at the neurula stage, one molecule each of 5S RNA and capped mRNA and 10 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.     

Polypeptide Elongation Factors of the Developing Chick Brain

Abstract: The polypeptide elongation factors (EF-1_L, EF-1_H, and EF-2) of the developing chick brain were separated and purified by means of a combination of gel chromatographic methods. The molecular weight of EF-1_H of the chick brain ranged from 5 to 10 × 10⁵, and was different from that of the chick liver (about 7 × 10⁵). The molecular weight of other purified factors was about 5 × 10⁴ for EF-1_L. and 9.4 × 10⁴ for EF-2. High activities of polyphenylalanine (poly-Phe) synthesis per mg protein in the developing chick brain were observed between the 3rd embryonic week and the 1st post-hatch week and declined afterwards. On the other hand, the levels of both EF-1 and EF-2 per mg protein in the brain were observed to be high in an early embryonic stage, gradually declining afterwards to the adult level. The brain EF-1_L was a major component of EF-1 in an early embryonic stage, while EF-1_H became recognizable in the 3rd embryonic week. Moreover, the EF-1_H activities were found to be more than double with regard to the binding reaction and to be more than 10-fold as active in respect to poly-Phe synthesis in comparison with the activities of EF-1_L. It is proposed that the brain EF-1_H could be due to aggregates consisting of EF-1_L, a stimulatory factor, and other components.     

Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification

A species-specific 16S rRNA oligonucleotide probe (ASRB1) was developed for the detection of Desulforhabdus amnigenus in anaerobic granular sludge. The presence of nucleic acids from cells of D. amnigenus in granular sludge was determined using ASRB1 as a specific primer for polymerase chain reaction (PCR) amplification or as a probe for dot blot hybridizations. The detection threshold and the reproducibility of these two methods were determined with sludge amended with 10⁴–10¹⁰ D. amnigenus cells per gram of volatile suspended solids (VSS). For D. amnigenus cells with a ribosomal RNA content of 15 fg cell⁻¹, the lowest number of target cells detected by hybridization was 1 × 10⁸ cells g⁻¹ VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 × 10⁷ g⁻¹ VSS. This corresponds to a threshold level for hybridization of 0·1–0·001‰ of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0·01–0·0001‰. The rRNA content of D. amnigenus was found to be affected by the growth rate and the growth phase, and it ranged from 19 fg cell⁻¹ in slow-growing cultures to 90 fg cell⁻¹ in fast-growing cultures. Therefore, the detection threshold of the dot blot hybridization method for fast-growing cells is lower than for slow-growing cells.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Multiple oral inoculations with Cryptosporidium parvum as a means of immunization for production of monoclonal antibodies

Abstract Oral immunization of suckling mice with Cryptosporidium parvum results in a humoral response to a limited set of antigens. Six-day-old BALB/c mice were each inoculated orally with 1 × 10⁶ viable oocysts and subsequently administered oral inoculations of 2 × 10⁶ viable oocysts at 30 and 60 days following the primary infection. After 45 days, mice were boosted with 1 × 10⁶ oocysts orally, plus soluble extracts equivalent to 2 × 10⁶ and 1 × 10⁶ oocysts given intravenously and intraperitoneally, respectively. Four days later, splenic lymphocytes were fused to Ag8 myeloma cells. Using this method, we have been able to select for monoclonal antibodies that predominately recognize sporozoite surface and apical complex antigens.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

Permeability of the Chara cell membrane for ethylene glycol, glycerol, meso-erythritol, xylitol and mannitol

The permeability of internodal cells of Chara australis R. Brown for polyol molecules was determined by using a turgor balance to measure the increase in the osmotic pressure of an internodal cell incubated in artificial pond water containing one of the polyol compounds tested. The permeabilities for ethylene glycol, glycerol, meso -erythritol, xylitol and mannitol were (4. 39 ± 0. 52) × 10⁻⁹, (1. 49 ± 0. 40) × 10¹⁰, (4. 92 ± 0. 27) × 10⁻¹⁰ (9. 9 ± 3. 4) × 10¹¹ and (7. 6 ± 4. 8) × 10⁻¹² m s⁻³, respectively. The permeability for glycerol was slightly smaller than that for meso -erythritol, whose molecular weight is larger than that of glycerol in this homologous series: but the reason for this is not clear.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes'milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 × 10² Enterobacteriaceae/ml, 3.9 × 10² coliforms/ml and 2.0 × 10² faecal coliforms/ml, whereas the respective mean counts were 6.2 × 10³/ml, 5.4 × 10³/ml and 1.3 × 10³/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Random mutagenesis of Legionella pneumophila with mini-Tn10

Abstract The degradation of sheets of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (BIOPOL^®) by aerobic sewage sludge was analyzed. Degradation of the polymer was highly dependent on the pH of the culture medium and was maximal between pH 7 and pH 8.5. Below pH 6 and above pH 9 the degradation rate was very low. Agitation of the culture fluid had relatively little influence on the rates of degradation. 1.2×10⁵ aerobic polymer-degrading bacteria per ml sewage sludge were identified by halo formation on solid poly(3-hydroxybutyrate) (PHB)-containing media. The number of PHB-degrading bacteria in other ecosystems amounted to 3.8×10³ per ml sludge of a fresh-water lake, 9.2×10⁵ per g garden-soil, 1.3×10⁶ per g field-soil and 4.3×10⁶ per g compost.     

Effect of Verticillium lecanii on biological characteristics and life table of Serangium japonicum (Coleoptera: Coccinellidae), a predator of whiteflies under laboratory conditions

Effects of entomopathogenic fungus Verticillium lecanii on biological characteristics and life table of Serangium japonicum , a predator of whiteflies against five different conidial concentrations (1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, and 1×10⁸ conidia/mL) were studied under laboratory conditions. The developmental periods for all immature stages (from eggs, 1st, 2nd, 3rd, 4th instar nymph and pupae up to emergence) among the treatments were significantly different when compared to that of control, and the longest development period was observed as treated with 1×10⁸ spore/mL. However, no significant difference on the percent survival of all immature stages was observed among the treatments and control. Also, there were no significantly different effects of V. lecanii on mean generation time, intrinsic rate, the finite rate of increase and longevity of S. japonicum among the treatments and control.     

Bacterial microflora in the gastro-intestinal tract of Dover sole (Solea solea L.), with emphasis on the possible role of bacteria in the nutrition of the host

Abstract There was a progressive increase in the size of the aerobic heterotrophic bacterial populations along the gastro-intestinal tract of farmed Dover sole. Moreover, higher counts were recorded in juvenile than in adult animals. Thus, in juvenile fish, 5.2 × 10⁵, 8.0 × 10⁵ and 9.8 × 10⁶ aerobic heterotrophs/g were recovered from the stomach/foregut, midgut and hindgut/rectum, respectively. In adult fish, comparative samples revealed the presence of only 3.0 × 10⁴, 7.0 × 10⁴ and 2.3 × 10⁵ bacteria/g, respectively. There bacteria were equated with Acinetobacter, Alcaligenes , Enterobacteriaceae representatives, Flavobacterium, Micrococcus, Photobacterium, Staphylococcus and Vibrio . Of the compounds tested, many isolates, particularly those recovered from the hindgut/rectum, degraded p -nitrophenyl- β - N -acetylglucosaminide, chitin and collagen. Consequently, it is likely that such organisms may contribute to nutritional processes within Dover sole.