Localization of gonadotrophic hormones in the dog pituitary gland

Summary Using the immunoperoxidase technique and antisera to the specific beta () subunits of FSH and LH¹, selective immunochemical staining was localized mostly in the same cell type in the pars distalis and pars tuberalis of the dog pituitary gland. However, some cells were consistently shown to react solely with antisera to either LH or FSH. The cells stained for FSH were at least 1.5 times less numerous than those shown to contain LH. In the pars distalis of adult male dogs the immunoreactive gonadotrophs varied greatly in their relative proportion and were mostly shown to be much less numerous than in bitches in the anestrus phase of the sexual cycle. These cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. The performic acid-alcian blue (pH 0.2)-PAS-orange G procedure stained the FSH/LH cells blue or turquoise, demonstrating TSH cells (blue-purple), ACTH/MSH cells (red-purple) and PRL cells (orange-red). The FSH/LH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not apply to gonadotrophic hormones, although some cells seem to be the source of either FSH or LH.Abbreviations for Pituitary Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin The authors are grateful to Dr. H. Wiemann for the statistical evaluation and to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistanceRecipient of a Research Scholarship from the Arabic Republic of Egypt     

Subcellular compartmentation of different lipophilic fluorescein derivatives in maize root epidermal cells

Summary Fluorescence microscopy offers some distinct advantages over other techniques for studying ion transport processes in situ with plant cells. However, the use of this technology in plant cells has been limited by our lack of understanding the mechanisms that influence the subcellular distribution of dyes after loading with the lipophilic precursors. In this study, the subcellular distribution of 5-(and 6-)carboxydichlorofluorescein (CDCF), carboxy-SNAFL-1, and carboxy-SNARF-1 was compared to that of 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) after incubation of maize roots with their respective lipophilic precursors. Previously, we reported that incubation of roots with BCECF-acetomethyl ester (BCECF-AM) led to vacuolar accumulation of this dye. Similar results were found when roots were incubated with CDCF-diacetate. In contrast, carboxy-SNAFL-1 appeared to be confined to the cytoplasm based on the distribution of fluorescence and the excitation spectra of the dye in situ. On the other hand, incubation of roots with carboxy-SNARF-1-acetoxymethyl acetate yielded fluorescence throughout the cell. When the cytoplasm of epidermal cells was loaded with the BCECF acid by incubation at pH 4 in the absence of external Ca, the dye was retained in the cytoplasm at least 3 h after the loading period. This result indicated that vacuolar accumulation of BCECF during loading of BCECF-AM was not due to transport of BCECF from cytoplasm to vacuole. The esterase activities responsible for the production of either carboxy-SNAFL-1 or BCECF from their respective lipophilic precursor by extracts of roots were compared. The characterization of esterase activities was consistent with the subcellular distribution of these dyes in root cells. The results of these experiments suggest that in maize root epidermal cells the subcellular distribution of these fluorescein dyes may be determined by the characteristics of the esterase activities responsible for hydrolysis of the lipophilic precursor.Abbreviations BCECF (BCECF-AM) 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (its acetoxymethyl ester) - BTB bis-trispropane - CDCF (CDCF-DA) 5-(and 6-)carboxy-2,7-dichlorofluorescein (its diacetate derivative) - DAPI 4,6-diamidino-2 phenylindole dihydrochloride - DMSO dimethylsulfoxide - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethane-sulfonic acid - SNAFL-1 (SNAFL-1-DA) carboxyl SNAFL-1 (its diacetate) - SNARF-1 (SNARF-1-AM) carboxyl SNARF-1 (its acetoxymethyl acetate)     

Sites of microtubule reorganization in tobacco BY-2 cells during cell-cycle progression

Summary The sites of microtubule (MT) reorganization were examined in synchronized tobacco BY-2 cells. The MTs of these cells were completely destroyed by a combined cold and drug treatment at 0 °C with 100 M propyzamide for 3 h. After the cells were washed and cultured at 30 °C, the reorganization of MTs was observed in detail. Sites for MT reorganization at each stage of the cell cycle were identified on the cell cortex and nuclei, the mitotic apparatus, the nuclei (or the nuclei and cell cortex), and the cell cortex in the S-G₂ phase, M phase, M/G₁ interface, and g₁ phase, respectively. The polypeptide synthesis elongation factor (EF)-1 is co-localized with these sites of MT reorganization. At some stages, microfilaments (MFs) were found to be involved in the reorganization of MTs. Based on these results, the mode of MT reorganization during cell cycle progression is discussed.Abbreviations EF-1 elongation factor 1 - MAP microtubule-associated protein - MF microfilament - MIs mitotic indices - MT microtubule     

Changes in ectonucleotidase activities in rat Sertoli cells during sexual maturation

Sertoli cell maturation is a complex process involving both morphological and biochemical changes. These cells have previously been shown to be targets for extracellular purine structures such as ATP and adenosine. These compounds evoke responses in rat Sertoli cells through the purinoceptor families, P₂X and P₂Y and PA₁. The signals to purinoceptors are usually terminated by the action of ectonucleotidases. In a previous work, we demonstrated that rat Sertoli cells have ecto-ATPdiphosphohydrolase (EC 3.6.1.5), ecto-5-nucleotidase (EC 3.1.3.5) and ecto-adenosine deaminase (ecto-ADA) (EC 3.5.4.4) activities. Here we investigated whether some changes occur during rat Sertoli cell maturation in these activities. Rat Sertoli cells obtained from rats of different ages representing the pre pubertal, mid pubertal and young adult (10-, 18- and 35-day-old, respectively) were cultured and used for different assays. The nucleotide hydrolysis was estimated by measuring the Pi released using a colorimetric method and by HPLC analysis. ATP and ADP hydrolysis was increased 3-fold during sexual maturation. AMP hydrolysis increased 4-fold in 10- to 35-day-old Sertoli cells. Similar results were obtained when we used other substrates to measure the extracellular hydrolysis of nucleotides (GTP, GDP, GMP and IMP). The ecto-ADA activity showed a 2-fold increase in the specific activity (18- to 35-day-old Sertoli cells). The termination of the purine cascade by adenosine degradation was faster in the 35- than in 18-day-old Sertoli cells. Follicle Stimulating Hormone (FSH) influences on the ectonucleotidase activities were investigated in 10- and 18-day-old Sertoli cells and a significant increase in the ATP and ADP hydrolysis was observed. Our results show an increase in the extracellular purine cascade during the Sertoli cell development, indicating a rise in the purine communication inside the seminiferous tubules with rat sexual maturation.     

Hydantoinase activity of immobilized non-growing Pseudomonas putida cells

Summary Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) activity of Pseudomonas putida DSM 84 was evaluated using cells immobilized in alginate beads and in a microporous hollow fibre bioreactor. Conversion of dihydrouracil into N-carbamyl--alanine was most efficient with alginate-immobilized cells. A 40 to 45% conversion was obtained in shake flasks and in continuous mode with packed bed columns. The highest volumetric productivity was obtained with a packed bed column operated at a dilution rate of 0.5 h^-1 (99 g of product. 100 l^-1 per hour). After 96 h the alginate beads began to swell and break apart; no free cells were detected however. Despite some initial loss of cells from the microporous hollow fibre bioreactor, a steady state was later established and maintained for 400 h at dilution rates of 0.1 and 0.25 h^-1.     

Purification and properties of trehalase in Frankia ArI3

Trehalase was purified from cultures of Frankia strain ArI3 grown on media with or without NH₄Cl. The purified enzyme was specific for trehalose, exhibited a broad pH optimum of pH 4.5 to 5.3 and had a K _m for trehalose of 4.2 mM. The trehalase was inhibited in vitro completely by sucrose, glucose and mannose and partially by mannitol and sorbitol. In addition to the specific trehalase, a mixture of non-specific - and -glucosidases which exhibited some activity with ,-trehalose as a substrate were also partially purified in Frankia extracts made from nitrogen-fixing cells. These enzymes were not detected in the purifications of crude extracts made from non-nitrogen-fixing cells (grown on media supplemented with NH₄Cl). Trehalase activity in crude extracts increased over time when cells were induced to fix nitrogen, and the maximum specific activity of trehalase from nitrogen-fixing cultures was 4 times the maximum activity from non-fixing cultures. Trehalase activity was also examined in crude extracts made from Frankia vesicle clusters isolated from Alnus rubra nitrogen-fixing nodules infected with ArI3. The maximum activity of trehalase in these clusters was 6–7 times greater than in the nitrogenfixing pure cultures of ArI3 and 26–33 times greater than the non-fixing pure cultures.Abbreviations pcv packed cell volume - DTE dithioerythritol - PMSF phenylmethylsulphonylfluoride - EDTA sodium ethylenediaminetetraacetate     

Tissue and subcellular immunolocalisation of enzymes of lignin synthesis in differentiating and wounded hypocotyl tissue of French bean (Phaseolus vulgaris L.)

Antisera raised againstl-phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and a cationic cell-wall peroxidase, which had all been purified from suspension-cultured cells of French bean, have been used to carry out immunogold localisations in the growing plant. Immunoglobulin-G fractions were prepared from each antiserum and used to study the distribution of the enzymes in differentiating and wounded hypocotyls by immunogold techniques and visualisation by both light and electron microscopy. Following silver enhancement to amplify the signal, proteins were detected by confocal microscopy in both developing (pre-xylem/ phloem) and later metaxylem stelar tissue.l-Phenylalanine ammonia-lyase and C4H also accumulated in cells adjacent to metaxylem, presumably involved in maintaining a supply of phenylpropanoid precursors to the enucleated xylem for further lignin synthesis. In these cells, PAL subunits were cytosolic although some were associated with endomembrane. Cinnamate-4-hydroxylase was wholly associated with membrane and particularly high concentrations were found in the Golgi bodies. The cationic peroxidase accumulated in xylem at sites of secondary thickening and in the middle lamella. The three proteins are also involved in defensive lignification. Thus when visualised by light microscopy, PAL and C4H were seen to accumulate to high levels throughout the cell types in wound sites and especially in the epidermal cells. An even more intense general distribution was found upon hyperinduction of wounded cells with-aminooxy--phenylpropionic acid. At the subcellular level, PAL was found to be localised in the cytosol in the wounded cells; however, because of the loss of membrane through mechanical damage, association with membrane structures, particularly endoplasmic reticulum, in unwounded cells is not entirely ruled out. Cinnamate-4-hydroxylase was associated with membranes when these were preserved. In wounded tissue, the peroxidase was found at the growing edges of tylose-like structures in the vascular xylem.Abbreviations AOPP -aminooxy--phenylpropionic acid - C4H cinnamic acid-4-hydroxylase - CHS chalcone synthase - GRP glycine-rich glycoprotein - HRGP hydroxyproline-rich glycoprotein - Ig immunoglobulin - PAL phenylalanine ammonia-lyase G.P.B. thanks the Agicultural and Food Research Council for support.     

Loss of microtubular orientation and impaired development of prophase bands upon inhibition of RNA synthesis in root meristem cells

Synchronous cells in Allium cepa L. root meristems were treated with 3deoxyadenosine (cordycepin, an inhibitor of RNA synthesis) when roughly half of the population had reached G₂ phase (fast cells) while the other half was still in late S (slow cells). Since this drug also blocks DNA replication, slow cells remained in S while the treatment was continued.Indirect immunodetection of tubulin showed that a band typical of prophase formed in some cells with a portion of their nuclear DNA unreplicated. Most of the cells arrested in late S which had not developed a prophase band had a diminished number of cortical microtubules. Changes in their orientation (from transversal to oblique or longitudinal) occurred in roughly a cycle time (28 h).In the presence of 3deoxyadenosine, fast cells which had reached G₂ at the start of the treatment proceeded to prophase and remained there. Mature prophase bands were also formed in these cells but eventually they disoriented and, finally, disappeared. The data suggest that microtubular orientation in the meristematic cells depends on long-lived RNA species.Abbreviations 3AdR 3deoxyadenosine - MT microtubule - PB tubulin band typical of prophase     

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [³H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 g·ml^-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 g·ml^-1 5-aminouracil and 40 g·ml^-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.Abbreviations APC aphidicolin - 5-AU 5-aminouracil - DAPI 4, 6-diamidino-2phenylindole - HU hydroxyurea - MI mitotic index - MT microtubule - PMSF phenylmethyl-sulfonyl fluoride - PPB preprophase band - %PPB percentage of cells with PPBs     

Gene transfer by electroporation into intact scutellum cells of wheat embryos

Summary Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-F capacitor and with 100 g DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MES 2-N-morpholinoethane sulfonic acid     

Electron microscopic cytochemical localization of nucleoside phosphatases in normal and chronic granulocytic leukaemic human neutrophils

Summary Using electron microscope cytochemistry and cells separated on Ficoll-Hypaque, Mg²⁺-dependent ATPase, ADPase and 5-nucleotidase were predominantly localized as ectoenzymes on normal human granulocytes. Large deposits of ATPase final reaction product and more finely granular deposits of 5-nucleotidase final reaction product were firmly attached to the outer surface of cell plasma membranes. The final reaction product from ecto-ADPase was, however, only loosely associated with the plasma membrane. In addition, finer deposits of ADPase final reaction product were seen in specific granules and in background cytoplasm. No nucleotidase phosphatase activity was localized to the alkaline phosphatase-containing granules (phosphasomes) recently described by Rustinet al.In granulocytes from patients with chronic granulocytic leukaemia, ecto-ATPase had a patchy distribution on the plasma membranes. There was considerable heterogeneity between cells with regard to ADPase and 5-nucleotidase localization. In some cells, ADPase was seen only as an ectoenzyme and in a few it was present in specific granules, but in others it was seen at both sites, while in some cells no activity was detected. 5-Nucleotidase localization was normal in some cells but lacking from many. No correlation was found between enzyme heterogeneity and the degree of morphological cell maturity.     

The human Ia system: Definition and characterization by xenogeneic antisera

The production of xenogeneic anti-Ia serum against Ia antigens in serum has been previously described in the mouse and we now describe the production of xenogeneic anti-human Ia antisera using similar methods. With an indirect resetting technique, Ia-like antibodies were shown to react with the majority of B cells (95%), a subpopulation of T cells, with carbonyl iron adherent cells, and with some E^–Ig^– null cells, but there was no reaction with red cells and platelets. These reactions were the same as those obtained with DRW antisera using cytotoxicity testing. In addition, antigens detected with xenogeneic antisera were also found in serum, where they were found to exist in a low molecular weight, dialyzable form. By the selective removal of different cell surface markers by cocapping, no association could be found with the specifities detected with the xenogeneic anti-Ia antisera and with surface Ig, ₂-microglobulin, or HLA-A and B specificities. Alloantibodies to DRW specificities (but not HLA-A, B specificities) were able to specifically block the binding of the rabbit anti-Ia antibodies to B cells, and reciprocal blocking of rabbit antisera by DRW antibodies was also observed. Several xenogenic antisera were produced by immunizing rabbits with the serum of different individuals. Each antiserum was shown to contain a number of different specificities, as they gave different reaction patterns with different individuals when testing was done both directly and by absorption. These xenogeneic anti-la sera also segregated in a family with HLA-A and B specificities. The detection of a polymorphic antigenic system segregating with the HLA complex, distinct from HLA-A and B specificities, and whose antigens occur predominantly on B cells is therefore described. Because of the similarity of the reactions of the xenogeneic antisera in man to those found in the mouse, and because of the close relationship to the DRW specificities, the system has been provisionally called the H.Ia system.Abbreviations used in this paper AET 2-aminoethyl isothiouronium bromide - ₂-M -2 microglobulin - BSA Bovine serum albumin - H.Ia Human Ia - HuRBC Human red blood cells - Ig Immunoglobulin - Ir Immune response - MHC Major histocompatibility complex - MLR Mixed lymphocyte reaction - NHS Normal human serum - NMS Normal mouse serum - PBL Peripheral blood lymphocytes - PBS Phosphate-buffered saline - RAHIg Rabbit anti-human immunoglobulin - RASIg Rabbit anti-sheep immunoglobulin - RFC Rosette-forming cells - SAHIg Sheep anti-human immunoglobulin - SARIy Sheep anti-rabbit immunoglobulin - SRBC Sheep red blood cells     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

Water droplets in intercellular spaces of barley leaves examined by low-temperature scanning electron microscopy

Low-temperature scanning electron microscopy was used to examine fracture faces in leaf blades taken from well-watered or drought-stressed barley (Hordeum vulgare L. cv. Mazurka) seedlings. The leaf blades were freeze-fixed while hydrated and were examined with or without gold-coating. There were droplets (with a smooth surface at the resolution achieved) on the surface of cell walls in leaf blades (0.91 g^-1 water content) from well-watered seedlings grown in an environment of 67% relative humidity. These were mainly on the vascular bundle sheath, the guard and subsidiary cells, and on some mesophyll cells around the substomatal cavity and between the stoma and vascular bundle. The droplets occurred, more abundantly, in the same places in seedlings from 100% relative humidity. They occurred on a few guard cells from wilting leaf blades (0.81 g·g^-1 water content) and were absent from severely drought-stressed leaf blades (0.15 g·g^-1 water content). The droplets sublimed at the same moment as both water which was in leaf cells and water which was allowed to condense (after freeze-fixation) on the wall surface. It is suggested that the droplets are aqueous. Their possible origin and importance is discussed.     

Possible role of cyclic AMP in the synthesis of chlorophyll in Chlorella fusca

The intracellular concentration of cAMP in the green alga Chlorella fusca was in the range of 2 · 10^-9 to 10^-8 moles/g dry weight and was strongly dependent on the growth conditions. The cAMP level was high with high light intensity, low nitrate or glucose concentration. Intracellular cAMP increased only by factor of 2 when high amounts (up to 10^-3 M) of cAMP were added to the medium. Most of the given cAMP was converted to 5-AMP.Addition of cAMP had little effect on the chlorophyll content of the cells, only at 10^-6 M some enhancement in photoautotrophic cultures was observed. On the other hand high amounts of cAMP in the medium increased the growth rate. DBcAMP^* showed a positive effect on chlorophyll synthesis and growth rate at much lower concentrations compared to cAMP.Stimulation effects of exogenous cAMP on the synthesis of chlorophyll were also observed in mixotrophic cultures with a high glucose/nitrate ratio, conditions where chlorophyll synthesis is repressed. Similar to autotrophic conditions DBcAMP was more effective than cAMP.These data indicate that cAMP may act in a system controlling the chlorophyll content of the cells in response to nutrients or light.Abbreviation DBcAMP^* N⁶-2-O-dibutyryl-adenosine-35-monophosphate     

Ultrastructure and enzyme histochemistry of the pancreatic islets in the horse

Summary The characteristic localization of the silver-negative A₂ cells in the central part of the pancreatic islets in the horse offers a good opportunity to study the ultrastructure and histochemistry of this type of islet cell. Electron microscopical analyses revealed that the A₂ cells contained dense spherical granules varying considerably in size. Light and dark A₂ cells were identified. The presence of numerous secretory granules of very low density was the most conspicous feature of the B cells. These cells also showed considerable differences in density. A second type of peripheral islet cell was characterized by a very high content of mitochondria and ribosomes. These small islet cells contained tiny granules and are probably identical with the A₁ cells.Negative reactions for alkaline and acid phosphatases were obtained throughout the islet tissue, while a strong glucose-6-phosphatase activity was displayed by the peripheral cells. The diphosphopyridine and triphosphopyridine nucleotide diaphorase activities were high in the peripheral cells, considerably weaker reactions being noted in the A₂ cells. On the whole there was a low succinic dehydrogenase activity in the islet tissue with a somewhat weaker enzyme staining in the A₂ than in the peripheral cells. The reactions for glucose-6-phosphate dehydrogenase and lactic dehydrogenase were also less pronounced in the A₂ cells than in the intensely reacting peripheral cells.The following abbreviations are used DPN Diphosphopyridine nucleotide - DPND Diphosphopyridine nucleotide diaphorase - DPNH Diphosphopyridine nucleotide, reduced form - G-6-PD Glucose-6-phosphate dehydrogenase - LD Lactic dehydrogenase - MTT 3,5-diphenyl-2-(4,5-dimethylthiazol-2-yl)-tetrazolium bromide - Nitro-BT 2,2-di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-biphenylene)-ditetrazolium chloride - SD Succinic dehydrogenase - TPN Triphosphopyridine nucleotide - TPND Triphosphopyridine nucleotide diaphorase - TPNH Triphosphopyridine nucleotide, reduced form Supported by the Swedish Medical Research Council and the research grant A-5759 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.     

Metabolism of p-fluorophenylalanine in p-fluorophenylalanine sensitive and resistant tobacco cell cultures

The metabolism of D- and L-p-fluorophenylalanine (PFP) in DL-PFP resistant and sensitive tobacco cell cultures (Nicotiana tabacum), cell lines TX4 and TX1, respectively, has been compared. The amino acid analogue was taken up at a lower rate by the resistant cell line TX4. Incorporation of PFP into protein was also considerably reduced in TX4 cells, compared to TX1 cells. This, however, resulted mainly from a diminished availability of PFP due to a more rapid conversion of PFP by TX4 cells. TX1 cells and TX4 cells converted PFP qualitatively in the same way. The only detectable metabolite of D-PFP was N-malonyl-D-PFP, while all metabolites of L-PFP were identified as sequent products of the initial deamination of L-PFP by the enzyme phenylalanine ammonia-lyase (PAL). As TX4 cells were endowed with higher PAL-activity than TX1 cells, the resistant cells were able to metabolize L-PFP more rapidly to give, e.g., p-fluorocinnamoyl glucose ester and p-fluorocinnamoyl putrescine. In the presence of the specific PAL-inhibitor -aminooxy--phenylpropionic acid TX4 cells were slightly more sensitive to PFP. This suggests that the better detoxification contributes to the acquired resistance. The use of PFP as specific indicator for cell lines with increased PAL-activity, and hence increased levels of phenolic compounds, is discussed.Abbreviations AOPP -aminooxy--phenylpropionic acid - MCW methanol:chloroform:water - PAL phenylalanine ammonia-lyase - PFP p-fluorophenylalanine - Phe phenylalanine     

Inorganic polyphosphates and heavy metal resistance in microorganisms

The mechanisms of heavy metal resistance in microbial cells involve multiple pathways. They include the formation of complexes with specific proteins and other compounds, the excretion from the cells via plasma membrane transporters in case of procaryotes, and the compartmentalization of toxic ions in vacuoles, cell wall and other organelles in case of eukaryotes. The relationship between heavy metal tolerance and inorganic polyphosphate metabolism was demonstrated both in prokaryotic and eukaryotic microorganisms. Polyphosphates, being polyanions, are involved in detoxification of heavy metals through complex formation and compartmentalization. The bacteria and fungi cultivated in the presence of some heavy metal cations contain the enhanced levels of polyphosphate. In bacteria, polyphosphate sequesters heavy metals; some of metal cations stimulate an exopolyphosphatase activity, which releases phosphate from polyphosphates, and MeHPO₄^? ions are then transported out of the cells. In fungi, the overcoming of heavy metal stresses is associated with the accumulation of polyphosphates in cytoplasmic inclusions, vacuoles and cell wall and the formation of cation/polyphosphate complexes. The effects of knockout mutations and overexpression of the genes encoding polyphosphate-metabolizing enzymes on heavy metal resistance are discussed.

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C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells

Summary The potential of tobacco BY-2 suspension-cultured cells for examining in vivo targeting and import of proteins into plant peroxisomes was shown recently in our laboratory. In the current study, the necessity and sufficiency of putative C-terminal targeting signals on cottonseed malate synthase and bacterial chloramphenicol acetyl-transferase (CAT) were examined in BY-2 cells. Cotton suspension cells also were evaluated as another in vivo peroxisome targeting system. Ultrastructural views of BY-2 cells showed that the peroxisomes were relatively small (0.1-0.3 m diameter), a characteristic of so-called unspecialized peroxisomes, Peroxisomes in cotton and tobacco cells were identified with anti-cottonseed catalase IgGs as distinct immunofluorescent particles clearly distinguishable from abundant immunofluorescent mitochondria and plastids, marked with antibodies to -ATPase and stearoyl-ACP 9 desaturase, respectively. The C-terminal ser-lys-leu (SKL) motif is a well-established peroxisome targeting signal (PTS 1) for mammals and yeasts, but not for plants. Antiserum raised against SKL peptides recognized proteins only in peroxisomes in cotton and tobacco cells. The necessity of SKL-COOH for targeting of proteins to plant peroxisomes had not been demonstrated; we showed that SKL-COOH was necessary for directing cottonseed malate synthase to BY-2 peroxisomes. KSRM-COOH, a conservative modification of SKL-COOH, was shown by others to be sufficient for redirecting CAT in stably-transformed Arabidopsis plants to the leaf peroxisomes. Here we show with the same CAT constructs (e.g., pMON316CAT-KSRM) that KSRM is sufficient for targeting transiently-expressed passenger proteins to unspecialized BY-2 peroxisomes. These results provide new direct evidence for the necessity of SKL-COOH (a type 1 PTS) and sufficiency of a conservative modification of the PTS 1 (KSRM-COOH) for in vivo, heterologous targeting of proteins to plant peroxisomes.Abbreviations CAT chloramphenicol acetyltransferase - CHO cells Chinese hamster ovary cells - DAB 3,3-diaminobenzidine - GUS -glucuronidase - ICL isocitrate lyase - KSRM lysine-serine-arginine-methionine - MS malate synthase - PBS phosphate-buffered saline - PTS peroxisome targeting signal - SKL serine-lysine-leucine - tobacco BY-2 Bright Yellow-2 Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Expression of the synaptic exocytosis‐regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction

Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2‐knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2‐knockout mice were significantly lower than those in wild‐type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons.