Sensitivity of whole body protein synthesis to amino acid administration during short-term bed rest

We tested the hypothesis that a reduced stimulation of whole-body protein synthesis by amino acid administration represents a major mechanism for the bed rest-induced loss of lean body mass. Healthy young subjects and matched controls were studied on the last day of a 14-day bed rest or ambulatory period, as part of the overall protocol "Short-term Bed Rest - Integrated Physiology" set up by the German Aerospace Centre (DLR) in co-operation with the European Space Agency. A balanced mixture of essential and non-essential amino acids was intravenously infused in the postabsorptive state for 3 hours at the rate of 0.1 g/kg/hour. The oxidative and non-oxidative (i.e., to protein synthesis) disposal of the infused leucine was determined by stable isotope and mass spectrometry techniques. The clearance of total infused amino acids tended to be greater (P=0.07) in the ambulatory group than in the bed rest group. When leucine clearance was partitioned between its oxidative and non-oxidative (i.e., to protein synthesis) components, the results indicated that the oxidative disposal was not statistically different in the bed rest and in the ambulatory groups. In contrast, the non-oxidative leucine disposal (i.e., to protein synthesis) was about 20% greater (P<0.01) in the ambulatory group than in the bed rest group. In conclusion, these preliminary data suggest that 14-day bed rest impairs the ability to utilise exogenous amino acids for protein synthesis.     

Effects of nicotine exposure on T cell development in fetal thymus organ culture: arrest of T cell maturation

There is evidence for both physiological functions of the natural neurotransmitter, acetylcholine, and pharmacological actions of the plant alkaloid, nicotine, on the development and function of the immune system. The effects of continuous exposure to nicotine over a 12-day course of fetal thymus organ culture (FTOC) were studied, and thymocytes were analyzed by flow cytometry. In the presence of very low concentrations of nicotine many more immature T cells (defined by low or negative TCR expression) and fewer mature T cells (intermediate or high expression of TCR) were produced. In addition, the numbers of cells expressing CD69 and, to a lesser extent, CD95 (Fas) were increased. These effects took place when fetal thymus lobes from younger (13-14 days gestation) pups were used for FTOC. If FTOC were set up using tissue from older (15-16 days gestation pups), nicotine had little effect, suggesting that it may act only on immature T cell precursors. Consistent with an increase in immature cells, the expression of recombinase-activating genes was found to be elevated. Nicotine effects were partially blocked by the simultaneous addition of the nicotinic antagonist d-tubocurarine. Furthermore, d-tubocurarine alone blocked the development of both immature and mature murine thymocytes, suggesting the presence of an endogenous ligand that may engage nicotinic acetylcholine receptors on developing thymocytes and influence the course of normal thymic ontogeny.     

Effect of exogenous flavonoids on nodulation of pea (Pisum sativum L.)

Selected flavonoids that are known as inducers and a suppressor of nodulation (nod) genes of the symbiotic bacterium Rhizobium leguminosarum bv. viciae were tested for their effect on symbiosis formation with garden pea as the host. A solid substrate was omitted from the hydroponic growing system in order to prevent losses of flavonoids due to adsorption and degradation. The presumed interaction of the tested flavonoids with nod genes has been verified for the genetic background of strain 128C30. A stimulatory effect of a nod gene inducer naringenin on symbiotic nodule number formed per plant 14 d after inoculation was detected at concentrations of 0.1 and 1 micro g ml(-1) nutrient solution. At 10 micro g ml(-1), the highest concentration tested, naringenin was already inhibitory. By contrast, nodulation was negatively affected by a nod gene suppressor, quercetin, at concentrations above 1 micro g ml(-1), as well as by another tested nod gene inducer, hesperetin. The deleterious effect of hesperetin might be due to its toxicity or to the toxicity of its degradation product(s) as indicated by the inhibition of root growth. Both the stimulatory effect of naringenin and the inhibitory effect of quercetin on nodule number were more pronounced at earlier stages of nodule development as revealed with specific staining of initial nodules. The lessening of the flavonoid impact during nodule development was ascribed to the plant autoregulatory mechanisms. Feedback regulation of nodule metabolism might also be responsible for the fact that the naringenin-conditioned increase in nodule number was not accompanied by any increase in nitrogenase activity. By contrast, the inhibitory action of quercetin and hesperetin on nodule number was associated with decreases in total nitrogenase activity. Naringenin also stimulated root hair curling (RHC) as one of the earliest nodulation responses at concentrations of 1 and 10 microg ml(-1), however, the same effect was exerted by the nod gene suppressor, quercetin, suggesting that feedback regulatory mechanisms control RHC in the range of nodulation-inhibiting high flavonoid concentrations. The comparison of the effect of the tested flavonoids in planta with nod gene activity response showed a two orders of magnitude shift to higher concentrations. This shift is explained by the absorption and degradation of flavonoids by both the symbionts during 3 d intervals between hydroponic solution changes. The losses were 99, 96.4, and 90% of the initial concentration of 10 micro g ml(-1) for naringenin, hesperetin, and quercetin, respectively.     

Extracellular matrix proteins influence phenotype and cytokine expression in human breast cancer cell lines

Tumor growth and invasion are not only the result of malignant transformation but are also dependent on environmental influences from surrounding stroma, extracellular matrix (ECM), local cytokines and systemic hormones. We have investigated the influence of ECM components on three human breast cancer cell lines of different malignant potential: MCF-7, T47D and MDA-MB-231 were cultured on collagen I, collagen IV, laminin, fibronectin or poly-D-lysine, and we analyzed the proliferation rate and cytokine expression pattern. Among the three cell lines investigated we observed a distinct response to each ECM component. We hypothesize that ECM may have a significant modulatory effect on malignant behavior in vivo which might depend on individual responses and on the differentiation state of tumor cells. This study also shows that the surface on which cells are cultured greatly influences cell kinetics and the cytokine expression pattern.     

Entry of Amphotropic and 10A1 Pseudotyped Murine Retroviruses Is Restricted in Hematopoietic Stem Cell Lines

Although transduction with amphotropic murine leukemia virus (MLV) vectors has been optimized successfully for hematopoietic differentiated progenitors, gene transfer to early hematopoietic cells (stem cells) is still highly restricted. A similar restriction to gene transfer was observed in the mouse stem cell line FDC-Pmix compared with transfer in the more mature myeloid precursor cell line FDC-P1 and the human erythroleukemia cell line K562. Gene transfer was not improved when the vector was pseudotyped with gp70^SU of the 10A1 strain of MLV, which uses the receptor of the gibbon ape leukemia virus (Pit1), in addition to the amphotropic receptor (Pit2). Although 10A1 and amphotropic gp70^SU bound to FDC-P1, K562, and fibroblasts, no binding to FDC-Pmix cells was detected. This indicates that FDC-Pmix cells lack functional Pit2 and Pit1 receptors. Pseudotyping with the vesicular stomatitis virus G protein improved transduction efficiency in FDC-Pmix stem cells by 2 orders of magnitude, to fibroblast levels, confirming a block to retroviral infection at the receptor level.     

Isolation of a Bacterial Strain with the Ability To Utilize the Sulfonated Azo Compound 4-Carboxy-4′-Sulfoazobenzene as the Sole Source of Carbon and Energy

A bacterial strain (strain S5) which grows aerobically with the sulfonated azo compound 4-carboxy-4′-sulfoazobenzene as the sole source of carbon and energy was isolated. This strain was obtained by continuous adaptation of “Hydrogenophaga palleronii” S1, which has the ability to grow aerobically with 4-aminobenzenesulfonate. Strain S5 probably cleaves 4-carboxy-4′-sulfoazobenzene reductively under aerobic conditions to 4-aminobenzoate and 4-aminobenzene-sulfonate, which are mineralized by previously established degradation pathways.It is generally assumed that sulfonated azo dyes are not degraded under aerobic conditions (14). Nevertheless, there have been some reports which suggest a conversion of certain sulfonated azo dyes under aerobic conditions (3, 7, 8, 13, 15). Furthermore, certain carboxylated analogs of sulfonated azo compounds are utilized aerobically as the sole source of carbon and energy by specifically adapted bacteria (11, 12, 16, 17). However, unequivocal evidence for the productive mineralization of a sulfonated azo compound by bacteria is lacking. In the present article the first observation of the utilization of a sulfonated azo compound as the sole source of carbon and energy by a bacterial strain is reported.Previously, a mixed bacterial culture which mineralizes sulfanilate (4-aminobenzenesulfonate) was isolated. This coculture consisted of the strains “Hydrogenophaga palleronii” S1 and Agrobacterium radiobacter S2 (4, 5). Because sulfanilate occurs as an azoaryl structural element in many azo dyes, it was of interest whether this mixed culture could adopt the ability to reduce azo bonds and release sulfanilate as growth substrate. Therefore, the model sulfonated azo compound 4-carboxy-4′-sulfoazobenzene (CSAB) was synthesized by nitro-amine condensation starting with sulfanilic acid and 4-nitrobenzoic acid (1). The precipitated CSAB was separated from the reaction mixture by filtration and purified by repeated dissolution in alkali and precipitation with acid. The identity and purity of the bright orange product were analyzed by UV-visible light spectroscopy, elementary analysis, and high-pressure liquid chromatography (HPLC). For the solid material obtained, molar extinction coefficients of 23.74 and 1.13 mM⁻¹ cm⁻¹ in water were determined at the wavelengths of 326 and 434 nm, respectively. The elementary analytic results were consistent with the structure of CSAB. The purity of the preparation was tested by HPLC with a reversed-phase column and a solvent gradient from 1 to 90% (vol/vol) methanol and 0.3% (vol/vol) H₃PO₄. A single band which showed absorbance at a wavelength of 326 nm was eluted. At 210 nm a minor contaminant (about 15% of the signal intensity of CSAB) was detected. This compound was clearly different from either 4-nitrobenzoate or sulfanilate.The mixed culture was grown in repeated batch cultures in a mineral medium with sulfanilate (5 mM). About every 2 weeks the culture was transferred (1:10 [vol/vol]) to fresh medium, in which the sulfanilate concentration was subsequently reduced and the CSAB concentration increased (±0.5 mM each). The color of the azo dye disappeared after 2 months. The culture was transferred to a solid mineral medium with CSAB as the sole source of carbon. From this culture was obtained strain S5, which grew aerobically with the sulfonated azo compound CSAB as its sole source of carbon and energy and with a doubling time of 9.5 h (Fig. ​(Fig.1).1). The complete disappearance of the dye was demonstrated by the loss of the orange color from the medium and by HPLC analysis, whereas CSAB was not degraded in a sterile control flask. Based on its colony morphology and the results obtained with the commercial identification system Biolog GN, this strain strongly resembled “H. palleronii” S1. Recently, it was demonstrated that, in the presence of low concentrations of biotin, cyanocobalamin, and 4-aminobenzoate, strain S1 also grows in axenic culture with sulfanilate (2). Therefore the adaptation experiment was repeated in the presence of these three substances with a pure culture of strain S1. This experiment also resulted in the isolation of a strain which grew in axenic culture with CSAB as the sole source of carbon and energy. Open in a separate windowFIG. 1Aerobic growth of strain S5 with CSAB as the sole source of carbon and energy. The growth was determined photometrically (OD₅₄₆), and the turnover of CSAB was measured by HPLC with a reversed-phase column and a solvent gradient consisting of H₂O, methanol, and 0.3% H₃PO₄ with increasing concentrations of methanol (1 to 90%). An OD₅₄₆ of 1 corresponded to 0.33 mg of protein ml⁻¹.To ensure that the genetic backgrounds of strains S5 and S1 were identical, the genes for the 16S rRNAs were amplified by PCR with different universal primers (6) and sequenced in comparison to the corresponding gene from the type strain, H. palleronii DSM 63. It was found that the sequences from strains S1 and S5 were > 99.8% identical (there were only two discrepancies between the two sequences), but they showed only 97.7 to 97.9% identity with the 16S rRNA gene from H. palleronii DSM 63. It was therefore concluded that strain S5 was derived from strain S1 and that the strains do not belong to the species H. palleronii.A reductive cleavage of the azo bond of CSAB would result in the formation of 4-aminobenzoate and sulfanilate. Like the parent strain, S1, strain S5 grew in the presence of sulfanilate, 4-aminobenzoate, and 4-sulfocatechol. The doubling times with these compounds were 6.2 to 6.4 h. We therefore investigated whether reductive cleavage of CSAB by strain S5 occurs. Strain S5 was grown aerobically with 5 mM CSAB, and cell extracts were prepared (10) in different buffers. These cell extracts were incubated aerobically in cuvettes containing 50 mM Tris-HCl buffer (pH 8.0), 0.5 mM CSAB, 1 mM NADH, or 1 mM NADPH and with various mixtures of possible cofactors. The enzyme activity was measured spectrophotometrically at the absorption maximum for CSAB (at a wavelength of 434 nm), but no significant decrease in absorbance was observed. Neither addition of a membrane fraction nor performing the enzyme assays under anaerobic conditions (9) improved the turnover of CSAB in the cell-free system. Furthermore, there was no significant increase in azo reductase activity when harvested cells were resuspended in the culture supernatant instead of Tris-HCl buffer.The maximal enzyme activities observed for cell extracts were only about 30% of the activities found for intact cells. This suggested that during the disruption of the cells some important components of the azo reductase system were destroyed or some cofactors were present in only limiting quantities.Because it was difficult to obtain reproducible enzyme activities with cell extracts, the turnover of CSAB by resting cells was investigated. Cells of strain S5 were grown with CSAB (5 mM), harvested by centrifugation, resuspended in Tris-HCl at an optical density at 546 nm (OD₅₄₆) of 5.3, and incubated in a water bath shaker (140 rpm; 30°C) with 0.5 mM CSAB (Fig. ​(Fig.2).2). Thus, the transient accumulation of two metabolites in the supernatants was observed by reversed-phase HPLC (column size, 250 by 4.6 mm) (SIL 100; Grom, Herrenberg, Germany). The solvent system consisted of a solvent gradient with increasing concentrations of methanol, starting with 1% (vol/vol) methanol, 98.9% (vol/vol) water, and 0.1% H₃PO₄. The flow rate was 0.7 ml min⁻¹. The metabolites formed were identified as sulfanilate and 4-sulfocatechol by comparison of their retention times and in situ UV-visible light-spectra with authentic standards. Surprisingly, the concentration of 4-sulfocatechol in the medium increased (and decreased) during the experiment more rapidly than the concentration of sulfanilate (Fig. ​(Fig.2).2). 4-Sulfocatechol also temporarily accumulated when resting cells of strain S1 were incubated with sulfanilate (4, 5). This suggested that in the resting-cell assay the initial activity of the sulfanilate-converting enzyme was higher than the activity of the 4-sulfocatechol-oxidizing enzyme protocatechuate-3,4-dioxygenase type II. Presumably, the activity of the sulfanilate-converting enzyme decreased during the experiment more rapidly than the activity of protocatechuate-3,4-dioxygenase type II. No accumulation of 4-aminobenzoate or protocatechuate was found by HPLC analysis during the experiment. In a control experiment with cells of strain S1 grown with 4-aminobenzenesulfonate, no turnover of CSAB was observed by HPLC analysis. Open in a separate windowFIG. 2Conversion of CSAB (•) to sulfanilate (▪) and 4-sulfocatechol (□) by resting cells of strain S5. Strain S5 was grown in a mineral medium with CSAB as the sole source of carbon and energy, and resting cells were prepared as described in the text.The detection of sulfanilate derived from CSAB suggested a reductive cleavage of CSAB, yielding sulfanilate as one of the reduction products. This reaction should also proceed in the absence of oxygen. Therefore, resting cells were incubated under anaerobic conditions with CSAB. Surprisingly, the rate of CSAB turnover under anaerobic conditions was <2% of the turnover rate under aerobic conditions.A further indication of a reductive cleavage of CSAB into sulfanilate and 4-aminobenzoate was obtained by growing strain S5 with CSAB or a complex medium (HPG medium) (4). When the cells were grown in a mineral medium with CSAB and the turnover of the substrates was analyzed by HPLC, it was found that resting cells converted CSAB, 4-aminobenzoate, or 4-aminobenzenesulfonate with specific activities of 0.012, 0.026, and 0.011 μmol min⁻¹ mg of protein⁻¹, respectively. In contrast, after growth of the cells in HPG medium, these activities were only 0.007, 0.010, and 0.003 μmol min⁻¹ mg of protein⁻¹, respectively. Incubation of resting cells with CSAB and different potential inhibitors of ring cleavage dioxygenases showed that the turnover of CSAB was almost completely inhibited by the addition of 8-hydroxyquinoline or 2,2′-bipyridyl (1 mM each). The presence of 4-nitrocatechol (0.25 mM) also resulted in a pronounced reduction of the rate of CSAB turnover (6% of the rate in the absence of the inhibitor). In this system as well the formation of 4-sulfocatechol was observed.The degradation of sulfanilate and 4-aminobenzoate by strain S1 has been previously studied (5). The proposed degradation pathway for CSAB and its reduction products is shown in Fig. ​Fig.3.3. Open in a separate windowFIG. 3Proposed pathway for the degradation of CSAB by strain S5. 4AB, 4-aminobenzoate; 4ABS, 4-aminobenzenesulfonate (sulfanilate); 3,4DHB, 3,4-dihydroxybenzoate (protocatechuate); 4SC, 4-sulfocatechol; 2H4CMSA, 2-hydroxy-4-carboxymuconic semialdehyde; 3SM, 3-sulfomuconate; 4SL, 4-carboxymethyl-4-sulfobut-2-en-4-olide (4-sulfolactone); MA, maleylacetate; 3OA, 3-oxoadipate; TCC, tricarboxylic acid cycle.To obtain some information about the substrate specificity, resting cells were incubated with CSAB, 4,4′-dicarboxyazobenzene (DCAB), 4-hydroxy-4′-sulfoazobenzene, methyl orange [4-(N,N-dimethyl)-4′-sulfoazobenzene; color index (C.I.) 13025], orange II {4-[(2-hydroxy-1-naphthalenyl)azo]-benzenesulfonic acid; C.I. 15510}, or sunset yellow FCF {6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthol-6-sulfonic acid; FD&C no. 6; C.I. 15985}. Of these compounds, only CSAB and DCAB were converted by resting cells. DCAB was also utilized by strain S5 as the sole source of carbon and energy. Furthermore, no growth of strain S5 was found with acid black 24 and 52, acid blue 113, acid red 1, amaranth, direct red 81, direct yellow 4 and 50, mordant yellow 3, and naphthol blue black.The results presented in this study suggest that bacterial cultures with the ability to aerobically degrade simple sulfonated azo dyes may be obtained after preadaptation to sulfonated aminoaromatics and/or when reductive cleavage of the azo bond gives rise to an aerobically assimilable aminoaromatic structure, like 4-aminobenzoate. This selection scheme circumvents the problems observed during attempts to adapt bacteria with the ability to degrade carboxylated azo compounds for the degradation of sulfonated azo compounds (12). The ability of strain S5 to mineralize CSAB suggests that it is possible to degrade sulfonated azo dyes under aerobic conditions if biological systems which can grow and can mineralize the reduction products are available.

Nucleotide sequence accession number.

The nucleotide sequences for the 16S rRNAs from strains S5 and S1 have been deposited in the GenBank data library under accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF019037","term_id":"3004497","term_text":"AF019037"}}AF019037 and {"type":"entrez-nucleotide","attrs":{"text":"AF019073","term_id":"3004495","term_text":"AF019073"}}AF019073, respectively.     

Remobilisation of vacuolar stored nitrate in barley root cells

Double-barrelled nitrate-selective microelectrodes have been used to measure the time course of the remobilisation of vacuolar stored nitrate in barley (Hordeum vulgare L. cv. Klaxon) root cells during 24 h of nitrate deprivation. These measurements showed that there are different time courses for this process in epidermal and cortical cells of the same root. The remobilisation was much slower from cortical cell vacuoles and had a time course which was similar to that obtained for tissue digests of the roots. The microelectrodes were also used to measure the nitrate concentration in sap exuding from detopped seedlings. These measurements showed that there was a gradual decrease in the delivery of nitrate to the shoot during this time. Root nitrate reductase activity of neither shoots nor roots changed significantly during the first 24 h. Direct measurement of the cytosolic nitrate in a root epidermal cell showed that during short-term changes, such as a 20-min exposure to zero external nitrate supply, cytosolic nitrate was maintained relatively unchanged. Net nitrate efflux from the roots was measurable during the initial 5 h of the zero-nitrate incubation period; after this time no further nitrate efflux was detectable. These measurements are discussed in relation to the nitrate budget of a root cell and we conclude that during the first 24 h of nitrate withdrawal vacuolar nitrate can be readily mobilised to supply the nitrogen demands of the seedling and to maintain the cytosolic nitrate concentration. Received: 31 July 1997 / Accepted 11 December 1997     

Interaction between photon flux density and elevated temperatures on photoinhibition in Alocasia macrorrhiza

The effects of light and elevated temperatures on the efficiency of energy conversion in PSII [?_PSII = (Fm′−Fs)/Fm′], pigment composition and heat tolerance of shade-acclimated Alocasia macrorrhiza were investigated. Leaf discs were exposed for 3 h to high light (HL; 1600 μmol photons · m⁻² · s⁻¹) or low light (LL; 20 μmol photons · m⁻² · s⁻¹) and a series of constant temperatures ranging from 30 to 49 °C. All HL treatments led to rapid and severe decreases in ?_PSII. During the 2-h recovery period (LL, 25 °C) following the HL treatments, fast and slow recovery phases could be distinguished. Leaf discs that had experienced HL and 30 °C recovered completely while no recovery of ?_PSII was seen after a 3-h exposure to HL and 45 °C. A 3-h exposure to 45 °C at LL led to a less severe decrease in ?_PSII and complete recovery was accomplished after less than 1 h. Under LL conditions a temperature of 49 °C was necessary to cause an irreversible decrease in ?_PSII, followed by necrosis the next day. Streptomycin had no effect on the degree of reduction and recovery in ?_PSII discs exposed to HL and 35–45 °C, but partially inhibited recovery in discs exposed to HL and 30 °C. Streptomycin led to a more severe decrease in ?_PSII at LL and 49 °C and completely inhibited recovery. Streptomycin had no effect on the conversion of the xanthophyll-cycle pigments during the treatment or the recovery. The epoxidation state was roughly the same in all leaf discs after a 3-h HL treatment (0.270–0.346) irrespective of the exposure temperature. The back-conversion of zeaxanthin into violaxanthin after a 2-h recovery period was only seen in leaf discs that had been exposed to HL and 30 °C. The thermotolerance of shade A. macrorrhiza leaves of 49.0 ± 0.7 °C (determined by fluorescence) coincided with the temperature at which damage occurred in leaf discs exposed to LL. However, under HL the critical temperature under which necrosis occurred was much lower (42 °C). The thermotolerance of A. macrorrhiza shade leaves could be increased by a short exposure (<20 min) to slightly elevated temperatures. Received: 11 June 1997 / Accepted: 9 September 1997     

Vinculin Is Part of the Cadherin–Catenin Junctional Complex: Complex Formation between α-Catenin and Vinculin

In epithelial cells, α-, β-, and γ-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. α-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell–matrix and cell–cell contacts, α-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell–cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and α-catenin. We show that α-catenin colocalizes at cell–cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH₂-terminal head binds to α-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The K_d of the complex was determined to 2–4 × 10⁻⁷ M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of α-catenin is involved in this interaction.