Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

Strontium induces murine keratinocyte differentiation in vitro in the presence of serum and calcium

Primary mouse keratinocytes in culture are induced to terminally differentiate by increasing extracellular Ca²⁺ concentrations (Ca_O) from 0.05 mM to ≥ 0.1 mM. The addition of Sr²⁺ (≥ 2.5 mM) to medium containing 0.05 mM Ca²⁺ induces focal stratification and terminal differentiation, which are similar to that found after increasing the Ca_O to 0.12 mM. Sr²⁺ in 0.05 mM Ca²⁺ medium induces the expression of the differentiation-specific keratins, keratin 1 (K1), keratin 10 (K10), and the granular cell marker, filaggrin, as determined by both immunoblotting and immunofluorescence. Sr²⁺ induces the expression of those differentiation markers in a dose dependent manner, with an optimal concentration of 5 mM. In the absence of Ca²⁺ in the medium, the Sr²⁺ effects are reduced, and Sr²⁺ is ineffective when both Ca²⁺ and serum are deleted from the medium. Sr²⁺ treatment increases the ratio of fluorescence intensity of the intracellular Ca²⁺ sensitive probe, fura-2, indicating an associated rise in the level of intracellular free Ca²⁺ and/or Sr²⁺. At doses sufficient to induce differentiation, Sr²⁺ also increases the level of inositol phosphates in primary keratinocytes within 30 min. The uptake curves of ⁸⁵Sr²⁺ by primary keratinocytes are similar to those of ⁴⁵Ca²⁺. At low concentrations, the initial uptake of both ⁴⁵Ca²⁺ and ⁸⁵Sr²⁺ reaches a plateau within 1 hr; at higher concentrations, the uptake of both ⁴⁵Ca²⁺ and ⁸⁵Sr²⁺ increases continuously for 12 hr. In keratinocytes pre-equilibrated with ⁴⁵Ca²⁺ in 0.05 mM Ca²⁺ medium, Sr²⁺ causes an increase of ⁴⁵Ca²⁺ uptake, which is dependent on the presence of serum. These results suggest that Sr²⁺ utilizes the same signalling pathway as Ca²⁺ to induce keratinocyte terminal differentiation and that Ca²⁺ may be required to exert these effects. © 1993 Wiley-Liss, Inc.     

Internode length in Pisum

The influence of the Na and Le genes in peas on gibberellin (GA) levels and metabolism were examined by gas chromatographic-mass spectrometric analysis of extracts from a range of stem-length genotypes fed with [¹³C, ³H]GA₂₀. The substrate was metabolised to [¹³C, ³H]GA₁, [¹³C, ³H]GA₈ and [¹³C, ³H]GA₂₉ in the immature, expanding apical tissue of all genotypes carrying Le. In contrast, [¹³C, ³H]GA₂₉ and, in one line, [¹³C, ³H]GA₂₉-catabolite, were the only products detected in plants homozygous for the le gene. These results confirm that the Le gene in peas controls the 3-hydroxylation of GA₂₀ to GA₁. Qualitatively the same results were obtained irrespective of the genotype at the Na locus. In all Na lines the [¹³C, ³H]GA₂₀ metabolites were considerably diluted by endogenous [¹²C]GAs, implying that the metabolism of [¹³C, ³H]GA₂₀ mirrored that of endogenous [¹²C]GA₂₀. In contrast, the [¹³C, ³H]GA₂₀ metabolites in na lines showed no dilution with [¹²C]GAs, confirming that the na mutation prevents the production of C₁₉-GAs. Estimates of the levels of endogenous GAs in the apical tissues of Na lines, made from the ¹²C:¹³C isotope ratios and the radioactivity recovered in respective metabolites, varied between 7 and 40 ng of each GA per plant in the tissue expanded during the 5 d between treatment with [¹³C, ³H]GA₂₀ and extraction. No [¹²C]GA₁ and only traces of [¹²C]GA₈ (in one line) were detected in the two Na le lines examined. These results are discussed in relation to recent observations on dwarfism in rice and maize.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Dual roles of CagA protein in Helicobacterpylori-induced chronic gastritis in mice

Cytotoxin-associated gene A (CagA) acts directly on gastric epithelial cells. However, the roles of CagA in host adaptive immunity against Helicobacter pylori (H. pylori) infection are not fully understood. In this study, to investigate the roles of CagA in the development of H. pylori-induced chronic gastritis, we used an adoptive-transfer model in which spleen cells from C57BL/6 mice with or without H. pylori infection were transferred into RAG2^−/− mice, with gastric colonization of either CagA⁺H. pylori or CagA⁻H. pylori. Colonization of CagA⁺H. pylori but not CagA⁻H. pylori in the host gastric mucosa induced severe chronic gastritis in RAG2^−/− mice transferred with spleen cells from H. pylori-uninfected mice. In addition, when CagA⁺H. pylori-primed spleen cells were transferred into RAG2^−/− mice, CD4⁺ T cell infiltration in the host gastric mucosa were observed only in RAG2^−/− mice infected with CagA⁺H. pylori but not CagA⁻H. pylori, suggesting that colonization of CagA⁺H. pylori in the host gastric mucosa is essential for the migration of H. pylori-primed CD4⁺ T cells. On the other hand, transfer of CagA⁻H. pylori-primed spleen cells into CagA⁺H. pylori-infected RAG2^−/− mice induced more severe chronic gastritis with less Foxp3⁺ regulatory T-cell infiltration as compared to transfer of CagA⁺H. pylori-primed spleen cells. In conclusion, CagA in the stomach plays an important role in the migration of H. pylori-primed CD4⁺ T cells in the gastric mucosa, whereas CagA-dependent T-cell priming induces regulatory T-cell differentiation, suggesting dual roles for CagA in the pathophysiology of H. pylori-induced chronic gastritis.     

Interspecies differences in the preference of ammonium and nitrate in vascular plants

Three solution experiments were performed to test the importance of NH ₄ ⁺ versus NO ₃ ^- +NH ₄ ⁺ to growth of 23 wild-forest and open-land species, using field-relevant soil solution concentrations at pH 4.5. At N concentrations of 1–200 M growth increased with increasing N supply in Carex pilulifera, Deschampsia flexuosa, Elymus caninus and Bromus benekenii. Geum urbanum was the most N demanding species and had little growth below 200 M. The preference for NH ₄ ⁺ or NO ₃ ^- +NH ₄ ⁺ was tested also at pH 4.0; no antagonism was found between NH ₄ ⁺ and H⁺, as indicated by similar relative growth in both of the N treatments at both pH levels. Growth in solution with NH ₄ ⁺ relative to NO ₃ ^- +NH ₄ ⁺ , 200 M, was negatively related to the mean pH of the field occurrence of the species tested; acid-tolerant species grew equally well with only NH ₄ ⁺ as with NO ₃ ^- +NH ₄ ⁺ (Oxalis acetosella, Carex pilulifera, Festuca gigantea, Poa nemoralis, Deschampsia flexuosa, Stellaria holostea, Rumex acetosella), while species of less acid soils were favoured by NO ₃ ^- +NH ₄ ⁺ (Urtica dioica, Ficaria verna, Melandrium rubrum, Aegopodium podagraria, Geum urbanum, Bromus benekenii, Sanguisorba minor, Melica ciliata, Silene rupestris, Viscaria vulgaris, Plantago lanceolata). Intermediate species were Convallaria majalis, Elymus caninus, Hordelymus europaeus and Milium effusum. No antagonism between NH ₄ ⁺ and Ca²⁺, Mg²⁺ and K⁺ was indicated by the total uptake of the elements during the experiment.     

Alterations in nitrogen assimilation and partitioning in nitrogen stressed plants

Although nutrient stress is known to alter partitioning between shoots and roots, the physiological basis for the phenomenon is unresolved. Experiments were conducted to examine assimilation of ¹⁵NO₃ by N-stressed plants and to determine whether apparent changes in assimilation in the root contributed to alterations in whole-plant partitioning of reduced-N. Tobacco plants (Nicotiana tabacum L. cv. NC 2326) were exposed to a low concentration of NO₃^? in solution (80 μM) for 9 days to effect a N-stress response. Exposure of plants to 1000 μM¹⁵NO₃^? for 12 h on selected days revealed that roots of N-stressed plants developed an increased capacity to absorb NO₃^?, and accumulation of reduced-¹⁵N in the root increased to an even greater extent. When plants were exposed to 80 or 1000 μM¹⁵NO₃^? in steady-state, ¹⁵NO₃^? uptake over a 12 h period was noticeably restricted at the lower concentration, but a larger proportion of the absorbed ¹⁵N still accumulated as reduced-¹⁵N in the root. The alteration in reduced-¹⁵N partitioning was maintained in N-stressed plants during the subsequent 3-day “chase” period when formation of insoluble reduced-¹⁵N in the root was quantitatively related to the disappearance of ¹⁵NO₃^? and soluble reduced-¹⁵N. The results indicate that increased assimilation of absorbed NO₃^?, in the root may contribute significantly to the altered reduced-N partitioning which occurs in N-stressed plants.     

Nitrate reductase and nitrate accumulation in relation to nitrate toxicity in Boronia megastigma

Moderate levels of N were toxic to the native Australian plant boronia (Boronia megastigma Nees). As NO^-₃ is the major N form available for plants under cultivated conditions, NO^-₃ reduction and accumulation patterns in boronia were examined following the supply of various levels of NO^-₃ to understand the physiological basis of this toxicity. At a low level of supplied NO^-₃ [15 mmol (plant)^-1], NO^-₃ was reduced without any detectable accumulation and without nitrate reductase activity (NRA) reaching its maximum capacity. When higher NO^-₃ levels [≥25 mmol (plant)^-1] were supplied, both NRA and NO^-₃ accumulation increased further. However, NRA increased to a maximum of ca 500 nmol NO^-₃ (g fresh weight)^-1 h^-1, both in the roots and leaves, irrespective of a 4-fold difference in the levels of supplied NO^-₃, whereas NO^-₃ continued to accumulate in proportion to the level of supplied NO^-₃. Chlorotic toxicity symptoms appeared on the leaves at an accumulation of ca 32 μmol NO^-₃ (g fresh weight)^-1. High endogenous NO^-₃ concentrations inhibited NRA. The low level of NRA in boronia was not limited by NO^-₃ or electron donor availability. It is concluded that the low NR enzyme activity is a genetic adaptation to the low NO^-₃ availability in the native soils of boronia. Thus, when NO^-₃ supply is high, the plat cannot reduce it at high rates, leading to large and toxic accumulations of the ion in the leaf tissues.     

Five novel elements involved in the regulation of mitosis in fission yeast

Summary Five new elements of the mitotic control in the fission yeast Schizosaccharomyces pombe were isolated from gene libraries as multicopy suppressors of the conditional lethal phenotype of win1-1 weel ^ts cdc25^ts triple mutant strains. These genes were designated wisl ⁺ –wis5⁺for win suppressing, and do not correspond to winl ⁺ or any of the previously characterised mitotic control genes. None of the wis genes is capable of suppressing the cdc phenotype of cdc25 ^ts strains, suggesting that their effect is not simply to reverse the effect of loss of cdc25 function. wisl ⁺ has been previously reported to encode a putative serine/threonine protein kinase that acts as a dosage-dependent inducer of mitosis. wis4 ⁺ appears to be a specific suppressor of the winl-1 mutation. wis2 ⁺ and wis3 ⁺ are capable of suppressing a wide range of cdc phenotypes arising from the combination of various mutations with wee1 ^ts and cdc25 ^ts, suggesting that the wis2 ⁺ and wis3 ⁺ products may interact with elements central to the mitotic control.     

Distribution of 46Sc and 51Cr in tumor-bearing animals and the mechanism for accumulation in tumor and liver

Tumor uptake rates, concentrations in mitochondrial fraction (containing lysosome) of liver and tumors, avid accumulations in connective tissue (especially inflammatory tissue) and binding substances in these tissues for ⁴⁶Sc³⁺ and ⁵¹Cr³⁺ were essentially similar to those for ⁶⁷Ga³⁺, ¹¹¹In³⁺, ¹⁶⁹Yb³⁺, ¹⁶⁷Tm³⁺, ⁹⁵Zr⁴⁺ and ¹⁸¹Hf⁴⁺. However, the main binding substance of ⁴⁶Sc³⁺ and ⁵¹Cr³⁺ in tumor and liver was the acid mucopolysaccharide (as described concerning ⁹⁵Zr and ¹⁸¹Hf) whose molecular weight exceeded 40,000, although the main binding substance of ⁶⁷Ga³⁺, ¹¹¹In³⁺, ¹⁶⁹Yb³⁺ and ¹⁶⁷Tm³⁺ was the acid mucopolysaccharide with a molecular weight of about 10,000.     

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic ^1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis ⁴. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects ⁵. For transmission between insect hosts, the bacteria colonize the intestine of the nematode''s infective juvenile stage ^6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects ^9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems ⁹.We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila¹⁴. Similar methods have been used to investigate other nematode-bacterium associations ^9,15-18and the approach therefore is generally applicable.The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization ^14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues ^14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication ²²or grinding ²³, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes ^21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization ^17,18, and is less laborious than other methods, including sonication ^22,25-27and individual nematode dissection ^28,29.     

Further observations on metabolic responses in hamster cells: changes in UDPG dehydrogenase activity.

The effects of phloretin, H₂DIDS (4,4′-diisothiocyano-1,2-diphenylethane-2,2′-disulfonate) and SO₄^?2 on anion transport in Ehrlich ascites tumor cells was studied in an effort to determine whether Cl^? and SO₄^?2 share a common transport mechanism. Sulfate, in the presence of constant extracellular Cl^? (100 mM), reduces Cl^? self-exchange by 43% (40 mM SO₄^?2) and Cl^??SO₄^?2 exchange by 36% (25 mM Cl^?/O SO₄^?2) compared to 25 mM Cl^?/50 mM SO₄^?2. Phloretin blocks without delay and to the same extent the self-exchange of both Cl^? and SO₄^?2. For example, at 10^?4 M phloretin, anion transport is inhibited 28% which increases to 78% at 5 × 10^?4 M. Reversibly bound H₂DIDS also inhibits the self-exchange of both Cl^? and SO₄^?2. However, at all H₂DIDS concentrations tested (0.5 ? 10 × 10^?5 M) SO₄^?2 transport was far more susceptible to inhibition than that of Cl^?. H₂DIDS when irreversibly bound to the cell inhibits SO₄^?2 but not Cl^? transport The results of these experiments are consistent with the postulation that both Cl^? and SO₄^?2 are transported by a common mechanism possessing two reactive sites.     

Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver

Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal^−/−:Soat2^+/+ mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs 1.9 mg in Lal^+/+:Soat2^+/+ littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal^−/−:Soat2^+/+ mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal^−/−:Soat2^−/− littermates. The level of EC accumulation in the SI of the Lal^−/−:Soat2^−/− mice was also much less than in their Lal^−/−:Soat2^+/+ littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal^−/−:Soat2^−/− mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function.     

Immune responses in vitro

The Mls locus was originally defined to have four alleles; three controlled products that were detectable in primary mixed leukocyte reactions (MLR), whereas one, b, was described as being null. Recently, other investigators postulated that the Mls locus is nonpolymorphic, being composed of the b null allele and of a singly expressed allele previously thought to be the a and d alleles. We previously reported that products controlled by Mls ^aand Mls ^dwere antigenically distinct and therefore are not controlled by the same allele, and the product of Mls ^bon cells of three different strains was easily detectable by Mls ^aand Mls ^dresponding cells. Thus the b allele is not null. In the present report evidence is presented which indicates that both Mls ^band Mls ^cencoded products were undetectable by MLR when in the presence of Mls ^aor Mls ^d. This was demonstrated by (a) the inability of Mls ^a/Mls ^cand Mls ^a/Mls ^bF₁ cells to stimulate Mls ^aresponding cells and Mls ^d/Mls ^cand Mls ^d/Mls ^bcells to stimulate Mls ^dcells; (b) the positive response of Mls ^a/Mls ^band Mls ^d/Mls ^bF₁-hybrid cells to Mls ^b-encoded products; and (c) the reactivity of Mls ^a/Mls ^cand Mls ^d/Mls ^cF₁ hybrid cells to Mls ^c-encoded determinants.     

Internode length in Zea mays L.

[¹³C, ³H]Gibberellin A₂₀ (GA₂₀) has been fed to seedlings of normal (tall) and dwarf-5 and dwarf-1 mutants of maize (Zea mays L.). The metabolites from these feeds were identified by combined gas chromatography-mass spectrometry. [¹³C, ³H]Gibberellin A₂₀ was metabolized to [¹³C, ³H]GA₂₉-catabolite and [¹³C, ³H]GA₁ by the normal, and to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₁ by the dwarf-5 mutant. In the dwarf-1 mutant, [¹³C, ³H]GA₂₀ was metabolized to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₂₉-catabolite; no evidence was found for the metabolism of [¹³C, ³H]GA₂₀ to [¹³C, ³H]GA₁. [¹³C, ³H]Gibberellin A₈ was not found in any of the feeds. In all feeds no dilution of ¹³C in recovered [¹³C, ³H]GA₂₀ was observed. Also in the dwarf-5 mutant, the [¹³C]label in the metabolites was apparently undiluted by endogenous [¹³C]GAs. However, dilution of the [¹³C]label in metabolites from [¹³C, ³H]GA₂₀ was observed in normal and dwarf-1 seedlings. The results from the feeding studies provide evidence that the dwarf-1 mutation of maize blocks the conversion of GA₂₀ to GA₁.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RP reverse phase     

Phytoplankton primary production in a mesotrophic pond in sub-tropical Bangladesh

Annual gross primary productivity in mesotrophic Shahidullah Hall pond (Dhaka, Bangladesh) was 1383.35 g C m⁻² y⁻¹ (arithmetic mean). Daily primary productivity (between 1.6 and 6.8 g C m⁻² d⁻¹ was correlated with chlorophylla, day length and dissolved silica. Chlorophylla related significantly withk, incident light, SRP, alkalinity and conductivity. A negative correlation existed between biomass and rainfall. Productivity, biomass, conductivity, alkalinity, and SRP increased after mid-winter.k, I _k andZ _eu varied according seasonally.P _max related directly with temperature. Seasonal variation of ∝ ^B was 0.0049–0.0258 mg C (mg chla mmol PAR)⁻¹ m⁻². Q₁₀ was 2.12, community respiration 1334.99 g C m⁻² y⁻¹, and the underwater light climate 186.43μE m⁻² s⁻¹.     

Cation-dependent uptake of zinc in human fibroblasts

The influence of K⁺ and Ca²⁺ on Zn²⁺ transport into cultured human fibroblasts was investigated. Zn²⁺ uptake was markedly reduced in the presence of both valinomycin and nigericin (electrogenic and electroneutral K ⁺ ionophores, respectively), and by reduction in the transmembrane K⁺ gradient produced by replacement of extracellular K⁺ with Na⁺, suggesting that Zn²⁺ may be driven by a Zn²⁺/K⁺ counter-transport system. To test the counter-transport hypothesis, we used ⁸⁶Rb as an analog of K ⁺ for efflux studies. The rate of Rb⁺ efflux was 3760 times that of Zn²⁺ uptake, thus the component of K⁺ involved in the Zn²⁺ counter-transport system was only a small proportion of the total K⁺ efflux. In investigating the effect of Ca²⁺ on Zn²⁺ uptake, we identified two components: (1) a basal Zn²⁺ uptake pathway, independent of hormonal or growth factors which does not require extracellular Ca²⁺ and (2) a Ca²⁺-dependent mechanism. The absence of Ca²⁺ decreased Zn²⁺ uptake, while increasing extracellular C+a²⁺ stimulated Zn²⁺ uptake. The effect was mediated by Ca²⁺ influx as the ionophores A23187 and ionomycin also stimulated Zn²⁺ uptake. We could not ascribe the Ca²⁺ effect to known Ca²⁺ influx pathways. We conclude that Zn²⁺ uptake occurs by a K⁺-dependent process, possibly by Zn²⁺/K⁺ counter-transport and that a component of this is also Ca²⁺-dependent.     

The Biosynthesis of Transfer Ribonucleic Acid in the Developing Rat Brain and in Cultured Glial Cells

Abstract: The biosynthesis of tRNA was investigated in cultured astroglial cells and the 3-day-old rat brain in vivo. In the culture system astrocytes were grown for 19 days and were then exposed to [³H]guanosine for 1.5–7.5 h; 3-day-old rats were injected with [³H]guanosine and were killed 5–45 min later. [³H]tRNA was extracted, partially purified, and hydrolyzed to yield [³H]-guanine and [³H]methyl guanines. The latter were separated from the former by high performance liquid chromatography and their radioactivity determined as a function of the time of exposure to [³H]guanosine. The findings indicate that labeling of astrocyte tRNA continued for 7.5 h and was maximal, relative to total RNA labeling, at 3 h, while in the immature brain tRNAs were maximally labeled at 20 min after [³H]guanosine administration. The labeling pattern of the individual methyl guanines differed considerably between astrocyte and brain tRNAs. Thus, [³H]1-methylguanine represented up to 35% of the total [³H]methyl guanine radioactivity in astrocyte [³H]tRNA, while it became only negligibly labeled in brain [³H]tRNA. Conversely, brain [³H]tRNA contained more [³H]N²-methylguanine than did astrocyte [³H]tRNA. Approximately equal proportions of [³H]7-methylguanine were found in the [³H]tRNAs of both neural systems. The [³H]methylguanine composition of brain [³H]tRNA was followed through several stages of tRNA purification, including benzoylated DEAE-cellulose and reverse phase chromatography (RPC-5), and differences were found between the [³H]methylguanine composition of RPC-5 fractions containing, respectively, tRNA^lys and tRNA^phe. The overall results of this study suggest that developing brain cells biosynthesize their particular complement of tRNAs actively and in a cell-specific manner, as attested by the significant differences in the labeling rates of their methylated guanines. The notion is advanced that cell-specific tRNA modifications may be a prerequisite for the successful synthesis of cell-specific neural proteins.     

The immunosuppressive tumor microenvironment in hepatocellular carcinoma

Increasing evidence indicates the immunosuppressive nature of the local environment in tumor. The present study was focused on analyzing the immune status within hepatocellular carcinoma. In contrast to the increasing number of CD4⁺ T cells, CD8⁺, CD3⁻CD56⁺, CD3⁺CD56⁺, and γδT cells were all found to be under-represented in tumor infiltrating lymphocytes. Notably, the relative abundance of CD3⁺CD56⁺ cells appeared to be correlated with patient survival. Functional analysis demonstrated that CD4⁺ cells in the tumor tended to produce more IL-10 but less IFN-γ, whereas CD8⁺ cells showed impaired capacity for the production of both IFN-γ and perforin. Consistent with previous reports, we observed a significant increase of Foxp3⁺ cells in the tumor tissue. Intriguingly, although over 90% of CD4⁺CD25^high cells were found to be Foxp3⁺, the majority of Foxp3⁺ cells were identified in the CD4⁺CD25^medium and CD4⁺CD25⁻ subsets. In support of its role as a negative regulator, CD4⁺CD25^high cells suppressed the proliferation of CD4⁺CD25⁻ cells isolated from the same tissues in an APC dependent manner. In conclusion, the tumor microenvironment of hepatocellular carcinoma is featured by the presence of multiple immunosuppressive factors.     

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.     

Differential superoxide dismutase expression in ryegrass cultivars in response to short term aluminium stress

Background and aims

Saline soils limit plant production worldwide through osmotic stress, specific-ion toxicities, and nutritional imbalances.

Methods

The ability of Ca²⁺ and K⁺ to alleviate toxicities of Na⁺ and Mg²⁺ was examined using 89 treatments in short-term (48 h) solution culture studies for cowpea (Vigna unguiculata (L.) Walp.) roots. Root elongation was related to ionic activities at the outer surface of the root plasma membrane.

Results

The addition of K⁺ was found to alleviate the toxic effects of Na⁺, and supplemental Ca²⁺ improved growth further in these partially-alleviated solutions where K⁺ was present. Therefore, Na⁺ appears to interfere with K⁺ metabolism, and Ca²⁺ reduces this interference. Interestingly, the ability of Ca²⁺ to improve K-alleviation of Na⁺ toxicity is non-specific, with Mg²⁺ having a similar effect. In contrast, the addition of Ca²⁺ to Na-toxic solutions in the absence of K⁺ did not improve growth, suggesting that Ca²⁺ does not directly reduce Na⁺ toxicity in these short-term studies (for example, by reducing Na⁺ uptake) when supplied at non-deficient levels. Finally, K⁺ did not alleviate Mg²⁺ toxicity, suggesting that Mg²⁺ is toxic by a different mechanism to Na⁺.

Conclusions

Examination of how the toxic effects of salinity are alleviated provides clues as to the underlying mechanisms by which growth is reduced.