THE UPTAKE OF γ-[3H]AMINOBUTYRIC ACID BY SLICES FROM VARIOUS REGIONS OF RAT BRAIN AND THE EFFECT OF LITHIUM

Abstract— Slices from various regions of rat brain, incubated at 25°C, rapidly accumulate [³H]GABA from the surrounding medium until after 60min tissue:medium ratios as high as 300 may be achieved. Kinetic analysis has demonstrated two distinct uptake systems for GABA in all the brain regions examined. One system has a relatively high substrate affinity ( K_m = 1.2 ± 10^-5 M) while the other has a lower affinity ( K_m = 4 ± 10^-4 M). Studies at low GABA concentration (5 ± 10^-8 M), as well as estimates of maximum velocities, have shown that the distribution of the high affinity uptake system is heterogeneous. Cortex, hypothala mus, midbrain and hippocampus have relatively high uptake rates while the striatum, cerebellum and pons and medulla have a lower uptake rate. Maximum velocities for the low affinity uptake system show much less regional variation.
Lithium, either added to the incubation medium or fed to rats, had no effect on the uptake of GABA by cortical slices.     

Triiodothyronine effect on some parameters of carbohydrate metabolism in rat blood and liver. I. In vivo effect of triiodothyronine on liver glycogen content, blood metabolites and serum glycogenolytic activity

Short (7 days) T3-treatment has no influence on the plasma glucose and FFA concentration and blood lactate level, as well as on the hepatic glycogen content in the rats. The rise in T3 levels, observed in our study, was accompanied by a fall in T4 concentrations, indicating suppression of the endogenous T4 production. On the other hand, glycogenolytic activity of rat serum in vitro is the highest in the system: control liver slices-serum of rats pretreated with T3 as compared to the system: control liver slices-control serum. These observations may lead to conclusions, that serum of rats pretreated with T3 contains factors exhibiting the greater ability to mobilize glucose from liver slices than the control serum (euthyroid). The possibility, that pretreatment with T3 may cause a decrease in the number and/or affinity of beta-adrenergic receptors in the rat liver, is discussed.     

Role of neuraminic acid in the heterogeneity of alkaline phosphatase in sheep brain

1. Comparative studies of the polyuridylic acid-directed phenylalanine-incorporating activity of cell-free systems derived from rat and chicken livers demonstrated markedly lower activity in the chicken liver system. 2. The chicken liver cell sap contained the factor(s) responsible for this lower activity. Ribosomes from chicken and rat performed equally well in the presence of rat liver cell sap. Chicken liver cell sap, when mixed with rat liver cell sap, caused an inhibition of incorporation of phenylalanine into acid-insoluble material. 3. Though ribosomal preparations and cell sap from both rat and chicken liver degraded polyuridylic acid to some extent, the chicken liver cell sap contained the largest amount of activity. 4. Rat liver cell sap inhibited the nuclease activities of ribosomal preparations, but no such nuclease inhibition could be demonstrated with chicken liver cell sap.     

Protein synthesis in the liver of rats injected with cholesteryl 14-methylhexadecanoate

1. Rats were injected intraperitoneally with cholesteryl 14-methylhexadecanoate and killed after various intervals of time up to 3 days; ribosomes and cell sap were isolated from their liver tissue. These fractions were tested for their ability to participate in protein synthesis. 2. Protein synthesis in complete systems containing ribosomes, cell sap and all necessary cofactors was significantly enhanced at 12 and 72h after the injection and significantly inhibited at 24h. At early times after injection isolated ribosomes had a slightly enhanced ability to bind nRNA. Peptide-elongation processes (i.e. binding of aminoacyl-tRNA to ribosomes, peptidyl transfer and polyphenylalanine synthesis) showed significant stimulation or inhibition depending on the time after injection of the ester. 3. A correlation was found between the ability of cell sap to stimulate polyphenylalanine synthesis and the relative cholesteryl 14-methylhexadecanoate content in the postmicrosomal supernatant at different time-intervals after administration of the ester. No significant changes were found in its content in the whole liver tissue. 4. Since the injected ester has previously been shown to accumulate in some enzymic fractions, the changes in its relative content may represent a regulatory mechanism modulating the rate of protein synthesis.     

Effects of flooding the root system of sunflower plants on the cytokin in content in the xylem sap

The severe chlorosis observed in the lower most of flooded sunflower plants (Helianthus annuus L. cv. Tall Single) may lie initiated by a reduction in the import of cytokinins by the stoot from the flooded root system. Experiment indicates that during 12 hours following the release of flooding, plants previously flooded for 72 hours or less recover their ability to exude sap when the root systems are aerated, and the root systems synthesize and export amino-acids to the shoot. Plants flooded for longer periods lose these abilities. The metabolic activity of the root apices declines parallel with the decline in eytnkinin concentration in the sap with increase in flooding time up to 72 hours. Flooding for 96 hours drastically reduce all four parameters of root activity. After flooding for this period there was a large increase in the number of blackened and tetrazolium-negative root apices which were in all probability dead. The correlation between the metabolic activity of the root apices and the total cytokinin content of the sap supports tbe view that root apices may be sites of cytokinin synthesis.     

Intercellular information transfer in the process of immunogenesis. IX. The induction of the synthesis of antiphage antibodies in lymphosarcoma cells of rats as affected by different fractions of immune nuclear RNA

Various fractions of the immune nuclear RNA were isolated from spleens of phage T2 immunized rats. The fractions were compared for their ability to induce anti-phage T2 antibody synthesis in transplantable lymphosarcoma cells. The most active proved to be the nuclear sap RNA and its subfraction with sedimentation constant of 10 S. The 4S and 26S subfractions RNA were less stable and in some experiments failed to induce antibody synthesis.     

Effect of cycloheximide on protein biosynthesis in rat liver

1. The liver ribosomes of rats given cycloheximide by intraperitoneal injection incorporate less amino acid into protein than ribosomes from control rat liver when they are incubated in vitro with excess of Sephadex-treated cell sap. The effect is rapid, marked and persistent. 2. Cell sap from liver of cycloheximide-treated animals is inhibitory but the inhibition can be relieved almost entirely by treating the cell sap with Sephadex. No damage has been done to the cell-sap factors: it is suggested that the dissolved cycloheximide in the cell sap causes the inhibition. 3. Cycloheximide added in vitro inhibits amino acid incorporation into protein in the presence or absence of polyuridylic acid. The inhibition is lessened by addition of excess of cell sap but is not abolished. 4. The differences between these results and those obtained with mouse liver (Trakatellis, Montjar & Axelrod, 1965) might arise because of species differences in sensitivity to the drug.     

Loss of heterozygosity at an unlinked genomic locus is responsible for the phenotype of a Candida albicans sap4Δ sap5Δ sap6Δ mutant

The diploid genome of the pathogenic yeast Candida albicans exhibits a high degree of heterozygosity. Genomic alterations that result in a loss of heterozygosity at specific loci may affect phenotypes and confer a selective advantage under certain conditions. Such genomic rearrangements can also occur during the construction of C. albicans mutants and remain undetected. The SAP2 gene on chromosome R encodes a secreted aspartic protease that is induced and required for growth of C. albicans when proteins are the only available nitrogen source. In strain SC5314, the two SAP2 alleles are functionally divergent because of differences in their regulation. Basal expression of the SAP2-2 allele, but not the SAP2-1 allele, provides the proteolytic degradation products that serve as inducers for full SAP2 induction. A triple mutant lacking the SAP4, SAP5, and SAP6 genes, which are located on chromosome 6, has previously been reported to have a growth defect on proteins, suggesting that one of the encoded proteases is required for SAP2 expression. Here we show that this sap4Δ sap5Δ sap6Δ mutant has become homozygous for chromosome R and lost the SAP2-2 allele. Replacement of one of the SAP2-1 copies in this strain by SAP2-2 and its regulatory region restored the ability of the sap4Δ sap5Δ sap6Δ mutant to utilize proteins as the sole nitrogen source. This is an illustrative example of how loss of heterozygosity at a different genomic locus can cause the mutant phenotype attributed to targeted deletion of a specific gene in C. albicans.     

Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation

Phytochelatins (PCs) are glutathione-derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long-distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long-distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four-fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long-distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long-distance source to sink transport of Cd as PC–Cd and glutathione–Cd complexes.     

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.     

Proton pump activity in bundle sheath tissues of broad-leaved trees in relation to leaf age

The fluorescent probe sulphorhodamine G (SR) has been previously used as an indicator of low extra-cellular pH and, by inference, of proton extrusion activity in living leaves. In legumes the SR uptake and proton extrusion was characteristic of the extended bundle sheath system (EBS) or paraveinal mesophyll, composed of bundle sheath cells and the related network of bridging cells between veins. This system has been identified as a site of temporary storage of amino carbon in soybean. A tree species. Populus deltoides Bartr. ex Marsh, was known both to have the EBS system in its leaves and to carry organic nitrogen in its xylem sap. It is now shown that P. deltoides also accumulates the SR probe in the EBS system. This association has been explored in 8 other broad-leaved tree species. Seven of the 8 species have EBS systems and accumulate SR in them in early summer. The 8th species, Tilia americana L. has no EBS system and shows weak SR accumulation. The capacity to accumulate SR (and by inference to scavenge solutes from the transpiration stream) disappeared in all species at various stages in late summer. In two species, in addition, SR accumulation is interrupted for several weeks during fruit growth. It is proposed that EBS systems will be found in many dicotyledonous leaves, and will be found to scavenge solutes, especially organic nitrogen, from the xylem sap.     

Protein profile of Lupinus texensis phloem sap exudates: Searching for Fe‐ and Zn‐containing proteins

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI‐MS and ESI‐MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19–21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe‐containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe‐binding proteins in phloem sap: a metallothionein‐like protein type 2B identified in the Fe‐affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem‐specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn‐binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins.     

Affinity driven molecular transfer from erythrocyte membrane to target cells

A wide variety of antimicrobial peptides are known to bind to - and disrupt microbial plasma membranes. Recently, derivatives of the antimicrobial peptide dermaseptin S4 were shown to selectively disrupt the plasma membrane of the intracellular parasite Plasmodium falciparum without harming that of the mammalian host cell. The resulting antimalarial activity is allegedly exerted after the harmless peptide binding to the membrane of the host cell, followed by peptide translocation across a number of intracellular membrane systems and interaction with that of the intraerythrocyte parasite. In this study, we present evidence in support of the ability of a membrane-bound peptide, the dermaseptin S4 derivative K(4)-S4(1-13)a, to transfer from red blood cells (RBCs) to another distant membrane. Binding of K(4)-S4(1-13)a to the plasma membrane of RBCs was assessed in vitro and in vivo, and found to be rapid, spontaneous and receptor independent, as was the transfer of the RBC-bound peptide to the plasma membrane of microorganisms. The present study further provides a basis for the possible use of RBCs as a transport vehicle to deliver drugs to distant targets. This drug delivery system involves the transient "loading" of RBCs with a lipophilic "hook" peptide. Such a peptide has enough affinity for the RBC's plasma membrane to bind to the membrane, but given the opportunity, the peptide will exit its position and transfer to another (target) cell for which it has a greater affinity. The efficacy of such an affinity driven transfer system was demonstrated experimentally by the transfer of K(4)-S4(1-13)a from pre-loaded RBCs to bacteria, yeast and protozoan target cells.     

Serological studies on sugar-beet yellows virus

Specific antisera have been prepared against the sap expressed from beet plants infected with beet yellows virus.
The antigen is unstable. In the sap it is destroyed by keeping for 2-3 days at room temperature or by heating for 10 min. at 52C. It is unaffected by p H changes between 5 and 9. In detached leaves at room temperature it remains unchanged for at least 6 days, whereas the ability of aphides to transmit from these leaves fell considerably in 4 days.
The antigen can be reversibly precipitated by ammonium sulphate or sedimented by high-speed centrifugation. However, all the attempts to isolate it from other sap constituents or concentrate it have failed.
The precipitin reaction is of value for diagnosis, and works successfully with crude sap.     

Reverse flow of sap in tree roots and downward siphoning of water by Grevillea robusta

1. Constant-power heat-balance sap flow gauges were used to compare sap flow in vertical and lateral roots of Grevillea robusta trees growing without access to ground water at a semiarid site in Kenya.
2. Reversal of sap flow occurred when root systems crossed gradients in soil water potential. Measurement of changes in the direction of flow was possible because of the symmetrical construction of the sap flow gauges; gradients in temperature across the gauges, and thus computed rates of sap flow, changed sign when reverse flow occurred.
3. Reverse flow in roots descending vertically from the base of the tree occurred, while uptake by lateral roots continued, when the top of the soil profile was wetter than the subsoil. The transfer of water downwards by root systems, from high to low soil water potential, was termed 'downward siphoning'; this is the reverse of hydraulic lift.
4. Downward siphoning was induced by the first rain at the end of the dry season and by irrigation of the soil surface during a dry period.
5. Downward siphoning may be an important component of the soil water balance where there are large gradients in water potential across root systems, from a wet soil surface downwards. By transferring water beyond the reach of shallow-rooted neighbours, downward siphoning may enhance the competitiveness of deep-rooted perennials.     

Virus Protein Synthesis in Animal Cell-Free Systems: Nature of the Products Synthesized in Response to Ribonucleic Acid of Encephalomyocarditis Virus

Ribonucleic acid (RNA) from encephalomyocarditis (EMC) virus stimulates the incorporation of amino acids into protein in cell-free protein-synthetic systems derived from Krebs mouse ascites tumor cells and chick embryo fibroblasts; the mouse system is the more responsive to the viral RNA. The greater part of this difference in activity can be ascribed to the cell sap, but the origin of the ribosomes also has a marked effect. The nature of the polypeptides formed in these cell-free systems was investigated by electrophoresis on polyacrylamide gels and by fingerprint analysis of tryptic digests. The same product in part appears to be synthesized in response to the EMC RNA in both systems. It was not detected if the EMC RNA was partly degraded (相似文献   

Thyroxine-binding proteins in liver and tail of Rana catebeiana undergoing metamorphosis.

Presence of a thyroxine-binding protein was demonstrated in vivo in cell sap of tail and liver of metamorphosing Rana catesbeiana tadpoles. Thyroxine-binding protein was not present in tail of prematamorphic tadpoles while it appeared during progressing metamorphosis roughly coinciding with the beginning of tail resorption. Susceptibility to pronase indicates that this thyroxine-binding macromolecule is protein in nature. Thyroxine-binding in liver was already present during premetamorphic stages and increased further during metamorphosis. A further difference between tail and liver thyroxine-binding protein was evidenced by molecular sieve chromatography on Sephadex G-200 indicating a molecular weight of thyroxine-binding protein in the tail of 60 000 as opposed to 42 000 for liver. Scatchard analysis of tail cell sap of tadpoles in metamorphic climax revealed a high affinity thyroxing binding site (Kd of 2 - 10(-10) M) of low capacity (1.7 pmol per mg protein) while tadpoles in premetamorphic stage had a thyroxine-binding site of lower affinity (9 - 10(-10) M) and higher capacity (4.8 pmol per mg protein). Thus affinity of thyroxine binding is 4-fold in metamorphic climax and appears to reflect the appearance of thyroxine binding observed in vivo.     

Spring Sap of Trees

In the spring, sap in perennial plants becomes available after winter dormancy. As is well known, substances including minerals and organic nutrients are transported from storage compartments in roots and stems to growth regions. For this solute distribution, xylem as well as phloem are used and probably also the cambial region and the ray system. In the present study, a puncture method has been used to record sap availability in six north European tree species during the spring. Attempts were made to withdraw sap from the trees at points 0.5 m, 1.3 m and 3.0 m above ground with “phloem needles” (constructed according to Hammel 1968), and a syringe. Sap could be withdrawn for the longest period for the sycamore (71 days). Birch (47 days) gave an intermediate period, beech (12 days), oak (12 days) and alder (13 days) gave short periods, and sap could not be withdrawn from ash. Effects of two environmental factors, temperature and light intensity on sap availability were examined. Temperature dependence of sap availability, already observed in the genus Acer, was confirmed in sycamore maple (Acer pseudoplatanus). There also seemed to be a correlation between sap availability and light intensity. Analysis of sugar in the tree sap revealed that sucrose was the only sugar in the sap of sycamore but hexoses (glucose and fructose) were preponderant in the sap of the other trees. The pH of sycamore sap (pH 6.9–5.4) fell as the season advanced. However, the pH-values of sap from the other trees did not vary significantly.     

Antioxidant defense enzymes in cell vacuoles of red beet roots

The vacuolar fraction isolated from red beet (Beta vulgaris L.) taproots was shown to contain the cyanide-sensitive Cu,Zn-activated superoxide dismutase (SOD; EC 1.15.1.1). The enzyme was represented by three isoforms located in the aqueous phase (in the vacuolar sap) without association to the membrane. Effective operation of SOD in plant cells, especially of its H₂O₂-sensitive molecular forms, is known to depend on peroxide-utilizing enzymes; this study revealed the existence of phenol-dependent peroxidase (EC 1.11.1.7) in the plant vacuoles. It was shown that the vacuolar peroxidase of red beet roots has a high affinity to benzidines and exhibits optimal activity at low pH (pH range 4–6 depending on substrate species). This peroxidase was represented by numerous molecular forms of acidic and basic nature. The isoenzyme composition of peroxidase in storage roots was highly labile: it depended on the duration of dormant period and comprised from 10 to 17 isoforms. The peroxidase isoforms were located both in the aqueous phase (vacuolar sap) and in the membrane, being weakly associated with the tonoplast. The presence of SOD and peroxidase in the vacuolar sap indicates the existence in vacuoles of an antioxidant defense system that protects vacuolar molecular structures against the impact of superoxide radicals and excessive amounts of H₂O₂.     

Cysteine metabolism in an isolate of Lactobacillus sake: Plasmid composition and cysteine transport

Abstract Lactobacillus sake L13, an isolate from vacuum packaged beef is able to utilise cysteine as an energy source for growth in rich medium. This isolate segregates into two strains, one of which retains the ability to grow on cysteine (notated as strong cells) while the other grows only marginally (weak). Both strains possess cysteine desulfhydrase activity, but only one is able to actively transport cysteine (high affinity). Uptake is sensitive to inhibition by valinomycin, CCCP, nigericin and arsenate. Comparison of the two cell types indicates that there are two cysteine transport systems and that the alternative one is passive and of low affinity. Strong cells possess two plasmids (2.7 and 8.3 kb) while the weak strain has lost the plasmids. Curing of the strong cells results in plasmid loss and loss of the high affinity cysteine uptake system.