Ion microscopic imaging of calcium during 1,25-dihydroxyvitamin D-mediated intestinal absorption

A combination of ion microscopic and conventional radionuclide techniques was employed to investigate the temporal-spatial dynamics of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]-stimulated intestinal calcium (Ca) absorption. At varying times following the administration of a single intravenous dose of 1,25(OH)₂D₃, to vitamin D-deficient chicks, transepithelial transport and tissue retention of Ca were quantitated in vivo, using the ligated duodenal loop technique and⁴⁷Ca as the tracer. The localization of Ca in the intestinal tissue during absorption was monitored by ion microscopy, using the stable Ca isotope,⁴⁴Ca, as the absorbed species. There was little transepithelial absorption of Ca in the vitamin D-deficient animals despite a substantial tissue accumulation of luminally derived Ca, the latter localizing predominantly in the brush border region of the enterocyte, as shown by the⁴⁴Ca-ion microscopic images. The early (30 min-1 h) response to 1,25(OH)₂D₃ was an increased tissue uptake of luminal⁴⁷Ca, which also primarily associated with the brush border region, again as shown by ion microscopy. At 2–4 h after the 1,25(OH)₂)D₃ dose, there was a progressive redistribution of Ca from the brush border region throughout the cytoplasm and into the lamina propria. At 8–16 h,⁴⁷Ca absorption was maximal and⁴⁴Ca was sparsely distributed in the intestinal tissue.⁴⁷Ca absorption gradually declined and reached pre-dose levels by 72 h. At this time, tissue⁴⁴Ca was again largely limited to the brush border region. These results provide support for the multiple actions of 1,25(OH)₂D₃ on the intestinal Ca absorption     

Selenoprotein inAspergillus terreus

Aspergillus terreus, a moderately selenium-tolerant fungus, metabolized⁷⁵ Se-selenite into several protein seleno-amino acids: selenomethionine and selenocysteine, as well as, nonprotein seleno-amino acids, selenocystathionine, and y-glutamyl selenomethyl selenocysteine. The results indicate the failure of the fungus to discriminate between sulphur and selenium. Selenium was also incorporated into several proteins of different molecular weights, mostly of low molecular weight proteins. Labeled studies showed the presence of high levels of selenomethionine and selenocysteine in the protein hydrolysate. The actual incorporation of protein selenoamino acids into the fungal protein was proven. The results demonstrated a finding that detracts from previous held views.     

Uptake of selenium-75 by PHA-stimulated lymphocytes

To determine which of a variety of inorganic and organic selenium compounds could best stimulate glutathione peroxidase, human lymphocytes were cultured with a number of selenium sources. The phytohemagglutinin-transformed lymphocytes were cultured in the presence of⁷⁵Se bound to serum proteins (25% v/v) or 10^?7 M concentrations of [⁷⁵Se]-selenite, [⁷⁵Se]-selenate, [⁷⁵Se]-selenocystine, and [⁷⁵Se]-selenomethionine. Organic forms of selenium were taken up in preference to inorganic forms. Control cultures, from which exogenous selenium had been omitted, showed a decreased level of glutathione peroxidase activity at the end of a 4 d culture period. Of the Se sources tested, [⁷⁵Se]-selenocystine and [⁷⁵Se]-labeled fetal calf serum proteins increased enzyme activity significantly, 79 and 47%, respectively, but selenite increased activity only by 7%. These results indicate that selenium from the two organic sources is most readily available for glutathione peroxidase synthesis.     

Chemical forms of selenium present in rat and ram spermatozoa

In vivo and in vitro studies were conducted to investigate the chemical forms by ion-exchange chromatography of selenium (Se) present in rat and ovine spermatozoa. After injection with ⁷⁵Se-selenite, the form of ⁷⁵Se in rat sperm was selenocysteine, but selenocysteine and selenomethionine (SeMet) were present in ovine sperm. Presumably, synthesis of SeMet by rumen microbes are responsible for its presence in ovine sperm. In vitro incubation of ram sperm with selenocysteine or SeMet produced no changes, but incubation with selenite produced a compound that eluted one fraction before SeMet from the ion-exchange column. After treatment of this fraction with mercaptoethanol, it eluted in a later fraction upon rechromatography, suggesting it to be selenodicysteine. This compound is apparently formed because of high levels of cysteine in semen. Cysteine, reduced glutathione, and oxidized glutathione were also found in semen. The significance of the results is discussed.     

Radioecological Risk Assessment of Low Selenium Concentrations through Genetic Fingerprints and Metabolic Profiling of Soil Bacterial Communities

In a context of environmental risk assessment of nuclear ⁷⁹Se radionuclide, the impact of low Se-selenite concentrations (0.008 and 8 mg kg⁻¹) on bacterial communities of two soils, a silty clay loam and a sandy soil, was investigated over a 6-month incubation time. This Se-selenite was partially labelled with ⁷⁵Se. The state of the Se-impacted bacterial communities was analyzed through total bacterial counts, DNA fingerprints (ARISA profiles) and metabolic profiling (carbon substrate utilization patterns). Furthermore, the genetic diversity of bacterial populations involved in Se volatilization was evaluated by tpm (thiopurine methyltransferase gene) profiling. Emissions of ⁷⁵Se and CaCl₂-extractable ⁷⁵Se were measured by γ-spectrometry and scintillation analysis. Se-selenite inputs changed transiently the substrate utilization patterns of bacterial communities but did not affect the other indicators. Se volatilization was at its highest level just after adding Se-selenite and for about 1 week. This volatilization was proportional to the added Se-selenite concentrations. It was 100-fold higher in silty clay loam, even though Se bioavailability was reduced in this soil. The soils were amended with crushed grass 3 months after the addition of Se-selenite. This organic amendment affected the organization of bacterial communities and increased the Se-volatilizing activities of both soils. Original soil organic carbon and bacterial diversity and activities seemed responsible for the different levels of Se emissions observed in soils. tpm lineages, encoding Se methyltransferases, were detected in both soils, confirming the broad distribution of tpm-harbouring bacteria and their probable role in the emissions of volatile Se. Five distinct groups of tpm were recorded per soil, with tpmI lineage being detected throughout the incubation period. This study demonstrates the ability of bacterial communities at volatilizing Se concentrations inferior to geochemical backgrounds and suggests that a probable transfer of nuclear Se will occur through volatilization after an environmental spill.     

Selenium-mediated biochemical changes in Japanese quails

The tissue uptake and distribution of injected [⁷⁵Se]-sodium selenite as a variance with time and as influenced by dietary selenium status was followed in the tissues of Japanese quails,Coturnix coturnix japonica. Quails maintained on a low selenium semipurified (basal) diet and basal diets supplemented with 0.2 and 2.0 ppm selenium as sodium selenite were injected intraperitonially with⁷⁵Se as sodium selenite (2.8 microcuries). The injected⁷⁵Se was monitored in blood, liver, kidney, heart, and testis at 24, 72, and 144 h after injection. Maximal uptake of the injected⁷⁵Se was observed in tissues of quails maintained on basal diet. The uptake of⁷⁵Se in tissues in general was determined by the dietary Se status. Among the organs studied, kidney had the maximal level of⁷⁵Se, 0.2 ppm (μg/g wet tissue) followed by liver, testis, and heart, but testis had the maximal level when the level per milligram of protein was considered, about 3.0 ng/mg protein, followed by liver, kidney, and heart. About 10–20% of the tissue⁷⁵Se was located in the mitochondria and 50–60% in the post-mitochondrial supernatant fractions in all dietary Se levels. Significant incorporation of⁷⁵Se in the mitochondrial membrane was observed. The percent distribution ratio between the membrane and matrix fractions of the mitochondria remained constant at all dietary Se levels which, in liver was 65∶35, in kidney 55∶45, and in testis 75∶25. However, in heart mitochondria, the distribution of⁷⁵Se between membrane and matrix varied with dietary Se status, the ratio being 82∶18 in the basal group, and 72∶28 and 41∶59 in the 0.2 and 2.0 ppm Se-supplemented groups, respectively. This is indicative of a preferential uptake of⁷⁵Se in the mitochondrial membrane in conditions of deficiency. About 40–60% of the mitochondrial membrane-associated⁷⁵Se was released upon Triton treatment in all the organs. Of the membrane-bound⁷⁵Se, about 10–15% was acid-labile in liver and kidney and 25% in the heart tissue. Possibilities of tissue specific roles, especially in the heart mitochondrial membrane-related processes, are indicated for selenium.     

Incorporation of Radioactive Seleno-(75Se)-Methionine into Mumps Virus

Mumps virus was grown in embryonated chicken eggs in the presence of radioactive seleno-(⁷⁵Se)-methionine. Virus in the allantoic and amniotic fluids was concentrated in a sucrose density gradient, and a peak of viral material coincided with a significant peak of ⁷⁵Se-radioactivity. The radioactivity was acid-insoluble and remained associated with the virus after purification by erythrocyte adsorption and elution and centrifugation on a second sucrose density gradient. After amino-acid hydrolysis of the radioactive virus, only ⁷⁵Se-methionine was recovered by chromatographic analysis. These results demonstrate that the radioactive ⁷⁵Se-methionine was incorporated into protein of infectious mumps virus.     

Probing the Structural Role of an αβ Loop of Maltose-binding Protein by Mutagenesis: Heat-shock Induction by Loop Variants of the Maltose-binding Protein that Form Periplasmic Inclusion Bodies

The maltose-binding protein (MBP) ofEscherichia coliis the periplasmic receptor of the maltose transport system. Previous studies have identified amino acid substitutions in an α/β loop of the structure of MBP that are critical for thein vivofolding. To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 ofmalEwere mutagenized. The maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correctin vivofolding pathway of the protein. By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies. Heat-shock induction with production of misfolded loop variants was examined using bothlon-lacZandhtrA-lacZfusions. We found that the extent of formation of inclusion bodies in the periplasm ofE. coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock σ factor, σ²⁴.     

Zinc-selenium interaction in the rat

Retention, dynamics of⁷⁵Se and⁶⁵Zn distribution, and elimination were studied in rats after separate or joint single doses of these metals. White female Wistar rats were divided into four groups (fifteen rats each). Group I received Na₂ ⁷⁵SeO₃ (0.1 mg Se/kg i.g.), group II received Na₂ ⁷⁵SeO₃+ZnCl₂ (5 mg Zn/kg s.c.), group III received⁶⁵ZnCl₂, and group IV received⁶⁵ZnCl₂+Na₂SeO₃. The zinc and selenium contents in the tissues were estimated during 120 h after administration; excretion in urine and feces of animals was determined throughout the experiment. Combined administration of zinc and selenium resulted in an enhanced selenium retention in the brain, spleen, kidneys, blood, lungs, and heart. A selenium-induced increase in the concentration of zinc was noted in the bowels, blood, liver, kidneys, spleen, brain, and lungs. The effects of the zinc/selenium interaction were visible especially in the lowered level of excretion of these elements. Zinc induced a decrease in the excretion of selenium in urine, with no concomitant changes in the excretion in feces. However, a visible decrease in the excretion of zinc in the feces was observed in the presence of selenium. The present results indicate an occurrence of clear-cut interaction effects between zinc and selenium administered simultaneously in the rat.     

Role of a protease in the adherence and enterotoxicity ofVibrio mimicus

The role of protease in the intestinal adherence and enterotoxicity ofVibrio mimicus strain E-33 was investigated by usingin vivo ligated rabbit ileal loop model.V. mimicus protease (VMP) was negative in fluid accumulation in the rabbit ileal loop. However, pre-treatment of the loop with purified VMP induced the elevation of fluid accumulation caused by the vibrios with the enhancement of bacterial adherence to the corresponding intestinal mucosa. The augmentation in the fluid accumulation could also be observed in the loop upon supplementation with VMP. In contrast, adherence to the mucosa was reduced by the simultaneous inoculation of VMP. The elevation in the fluid accumulation could also be observed in the loop supplemented or pre-treated with protease from V.cholerae orV. vulnificus. Furthermore, when the vibrios in the loops were accompanied by anti-VMP IgG antibody or inhibitors for VMP, such as ethyleneglycol-bis (\-aminoethylether)-N, N, N’, N’,-tetraacetic acid or tetraethylenepentamine, reductions in the adherence indices with consequent reductions in the fluid accumulations were observed. It is therefore, suggested that the protease produced by the pathogen contributes significantly toward the pathogenicity ofV. mimicus.     

Metabolism of zinc-binding ligands in rat small intestine

Studies were conducted to determine the effects of zinc deficiency and excess zinc intake on the relative⁶⁵Zn-binding activities of metallothionein (MT) and low-molecular-weight zinc-binding ligand (LMW-ZBL) in vitro and in vivo. Zinc-binding ligands of small intestine from four groups, each of five rats (normal, zinc-deficient, excess zinc injected, and excess zinc given orally), were separated by column chromatography on Sephadex G-75. The ratio of⁶⁵Zn binding activities of MT to LMW-ZBL (MT/LMW-ZBL) in zinc-deficient rats was decreased both in vitro and in vivo compared to the control. When excess zinc was administered orally,⁶⁵Zn-binding activity of MT was low in vitro and substantially increased in vivo. However, when excess zinc was injected intraperitoneally,⁶⁵Zn-binding activity of MT in vitro greatly increased, but⁶⁵Zn-binding activities of both MT and LMW-ZBL were significantly reduced in vivo as compared to the control. Based onA ₂₈₀ readings of isolated MT and densities of protein bands in disc gel electrophoresis, the⁶⁵Zn-binding activity of MT in vitro appeared to be proportional to the MT content. Hence, these data indicate that oral administration of excess zinc decreases MT whereas intraperitoneal injection of excess zinc stimulates its synthesis. Zinc deficiency has little to no effect on the intestinal MT metabolism. These results suggest that MT may be important in zinc secretion but not involved in zinc absorption; while LMW-ZBL participates both in zinc absorption and secretion.     

Serology and polymorphism ofH-2L- locus encoded antigens

Thirty B10.W congenic lines were analysed serologically, both by direct cytotoxicity and by absorption, for the presence of H-2L antigens. Three new H-2L antigens, 73, 74, and 75, were discovered. The B10.W lines and the inbred strains can be classified into at least six H-2L phenogroups on the basis of their reactivity withH- 2^dm2 anti-H- 2^d serum: BALB/c, B10.BUA1, B10.GAA37, B10.BUA16. B10.KPB128, and the negative group. Twenty-oneD-end recom-binants were analysed for the possible separation ofH-2D andH-2L loci. The failure to find such a separation indicates that theH-2D andH-2L loci are tightly linked. Serological analysis also indicated that theH- 2^dm1 has lost most of its H-2L antigens but retained at least one specificity which can be detected byH- 2^dm2 anti-H- 2^d serum.     

In situ patterns of nuclear replication in brain ganglia ofl(2) gl 4 mutant larvae ofDrosophila melanogaster

The1(2) gl ⁴ mutation (a deletion mutation of a recessive oncogene) ofDwsophila melanogaster causes neuroblastoma in the optic centres of brain of late third instar larvae. We have studied thein situ patterns of DNA synthesis in these brains by immunocytochemical detection of cells incorporating 5-bromodeoxyuridine. It was seen that1 (2)gl ⁴ brains from younger 3rd instar larvae had fewer replicating cells than in wild type larvae of comparable age but in brain ganglia of olderl (2) gl ⁴ larvae the number of replicating cells was much higher. The spatial distribution of replicating cells in optic lobes of brain ganglia of1 (2) gl ⁴ larvae was disturbed from early 3rd instar stage, much before the tumourous growth was morphologically detectable. The stereotyped pattern of asymmetrical cell divisions of the neuroblasts and their progeny cells was also not seen in1 (2) gl ⁴ brain ganglia. Therefore, it appears that thel (2)gl ⁴ product has an important role early in development to determine the temporal and spatial patterns of neuroblast cell division in developing brains.     

The metabolism of selenite and selenomethionine in mouse fibroblasts grown in tissue culture

Recent reports have provided evidence that selenium is an essential growth factor for cells grown in tissue culture. The aim of the work reported in this paper was to evaluate mouse fibroblasts as a model for the study of selenium metabolism in mammalian cells. The results showed that transformed mouse lung fibroblasts grown in media containing 9.1% bovine serum did not show a growth response to added selenium as selenite over the range of 10–1000 ng/mL. Uptake of selenium by cells was a direct function of the selenium concentration in the medium. The rate of uptake varied with the time of exposure of the cells to the selenium, and to the form of selenium in the medium. Experiments using radioactive selenium showed that⁷⁵Se from selenite was rapidly absorbed into the cell wall, but slowly incorporated into the soluble protein fraction.⁷⁵Se from selenomethionine was more slowly absorbed into the cells, but once inside, it became rapidly incorporated into soluble cytoplasmic proteins. Cell fractionation and gel filtration procedures established that⁷⁵Se from selenite was rapidly incorporated into glutathione peroxidase (GSHpx), whereas⁷⁵Se from selenomethionine was initially incorporated into a wide spectrum of proteins and only after a longer period did the⁷⁵Se peak become associated with GSHpx. These findings suggest fundamental differences exist in the manner in which mammalian cells initially absorb and metabolize different selenium compounds.     

Effects of glutathione and of cysteine on intestinal absorption of selenium from selenite

The influence of glutathione (1 mmol/L) (GSH) on in vitro mucosal uptake and in vivo absorption of⁷⁵Se-labeled selenite (10 μmol/L) was investigated in rat jejunum. For comparison, the effect ofl-cysteine (1 mmol/L) on in vivo absorption of⁷⁵Se-labeled selenite was also studied. In the in vitro, uptake experiments, only the mucosal surface was exposed to the incubation medium for 3 min. For the in vivo experiments, a luminal perfusion technique was employed. GSH inhibited in vitro mucosal Se uptake, whereas absorption in vivo was stimulated by GSH.l-Cysteine also stimulated in vivo Se absorption, confirming former in vitro mucosal uptake experiments. Thus, unlikel-cysteine, GSH affected in vitro and in vivo absorption of Se from selenite differently. Enzymatic cleavage of products of the reaction of selenite with GSH occuring more efficiently under in vivo than in vitro conditions may be a prerequisite for the stimulatory effect of GSH on Se absorption. This apparently does not apply to the stimulatory effect of cysteine. Since, GSH occurs in the intestinal lumen under physiological conditions, it may contribute to the high bioavailability of Se from selenite.     

Exclusion of selenium from proteins of selenium-tolerant astragalus species

Protein fractions from three selenium-tolerant and three selenium-sensitive Astragalus species, grown in the presence of [⁷⁵Se]selenate, were analyzed for their selenium content. Though tolerant species are known to accumulate considerably more selenium than do sensitive plants, protein fractions from the three selenium accumulators were found to contain significantly less selenium (0.46 to 0.57 picomoles selenium per milligram protein) than did protein fractions from the three nonaccumulators (4.17 to 5.02 picomoles selenium per milligram protein). Under similar conditions, seedlings of Vigna radiata (L.) Wilczek had taken up selenium (6.31 picomoles selenium per milligram protein) at levels comparable to those observed in the proteins of the nonaccumulator Astragali. These results establish that the ability to tolerate and to circumvent the toxic effects of selenium, characteristic of the accumulator species of Astragalus, is associated with a reduced incorporation of this element into protein.     

An eye imaginal disc-specific transcriptional enhancer in the long terminal repeat of thetom retrotransposon is responsible for eye morphology mutations ofDrosophila ananassae

Optic morphology (Om) mutations ofDrosophila ananassae are semidominant, neomorphic and nonpleiotropic, map to at least 22 loci scattered throughout the genome, and are associated with the insertion of thetom retrotransposon. Molecular and genetic analyses have revealed that eye morphology defects ofOm mutants are caused by the ectopic or excessive expression ofOm genes in the eye imaginal discs of third instar larvae. It is therefore assumed that thetom element carries tissue-specific gene regulatory sequences which enhance expression of theOm genes. In the present study, we examined whether or not the long terminal repeats (LTR) of thetom element contain such an eye imaginal disc-specific enhancer, usingD. melanogaster transformants containing alacZ gene ligated to thetom LTR. Analyses oflacZ gene expression in the eye imaginal discs of third instar larvae of 18 independently established transformant lines showed that thetom LTR was capable of enhancinglacZ expression in all the transformant lines, but the degree of enhancement varied between lines. In addition, the effect of thetom LTRlacZ gene evidently changed when thetom LTR construct was relocated to different chromosomal positions. On the basis of these findings, it is hypothesized that ectopic and excessive expression of theOm genes in the eye imaginal discs is induced by an eye imaginal disc-specific enhancer present in thetom LTR, the effect of which may be subject to chromosomal position effects.     

Reduced intestinal calcium and dietary calcium intake,increased aluminum absorption,and tissue concentration in the rat

To test the influence of calcium (Ca) on aluminum (Al) absorption, Ca was withheld from or added (1 mM) to the perfusate of thein situ rat gut. The rats had been maintained on Purina Rat Chow. Ca addition significantly decreased (to 70%) the rate of Al disappearance from the gut and decreased (to 55%) the area under the curve of Al appearance in portal blood. To test the influence of Ca deficiency on Al absorption, rats were maintained on a low-Ca (0.008%) or a Careplete (0.5%) diet for 1–4 wk. Thein situ gut was prepared, and a perfusate containing approximately 1 μM Ca was used. The rate of Al disappearance from the gut of low-Ca diet rats was significantly faster than from the gut of rats maintained on the Ca-replete diet, averaging 156% of the latter. Al appearance in portal blood was significantly greater (averaging 38%) in rats maintained on the low-Ca diet than in controls. To determine if Ca deficiency influences Al tissue distribution independent of gastrointestinal Al absorption, rats maintained on a low-Ca or a Ca-replete diet received 20 ip Al injections over 1 mo. Rats eating the low-Ca diet demonstrated enhanced tissue Al accumulation in all tissues studied, except for muscle and cerebral cortex. These results demonstrate enhanced Al absorption and tissue retention in the presence of reduced intestinal Ca concentration and reduced Ca intake.