Structural and Functional Rearrangements of Mesophyll as a Probable Basis for the Cytokinin-Dependent Assimilate Translocation in Detached Leaves

The effects of benzyladenine (BA) on the mesophyll functioning, such as osmotic potential (), the effect of the inhibitors of ⁺-ATPase on the influx of ¹⁴C-sucrose, the direction of carbon metabolism, and the rate of dark respiration, were followed in the detached leaves of pumpkin (Cucurbita pepo L.) and broad beans (Vicia faba L.). BA elevated and established a gradient of (_p) between the treated and untreated leaf regions. The inhibitors of H⁺-ATPase did not affect the BA-induced influx of ¹⁴C-sucrose. The changes were accompanied with the elevated synthesis of starch and other polymeric compounds and the diminished synthesis of the substances of relatively low molecular weight. The stimulation of dark respiration was short and inconsiderable. The author concludes that the BA-induced transport was a passive process related to a increase. Leaf expansion accompanied by the synthesis of high-molecular-weight substances essential for cell growth and by starch synthesis apparently increased the sink capacity of the BA-treated detached leaves. The diminished efflux from the leaf blade was probably related to a lowered level of the transportable carbon compounds restricting their entry into the phloem. The influx induction could result from the activation of growth and metabolic processes, the decline in the number of organic molecules per cell volume unit, and the development of _p between the source and sink leaf regions.     

The αβ complexes of ATP synthase: the α3β3 oligomer and α1β1 protomer

The basic structures of the catalytic portion (F₁, ₃₃) of ATP synthase are the ₃₃ hexamer (oligomer with cooperativity) and ₁₁ heterodimer (protomer). These were reconstituted from the and subunits of thermophilic F₁ (TF₁), and the ₃₃ hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F₁ and TF₁, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F₀F₁) is driven by an electrochemical potential of H⁺ (H⁺). F₀F₁ is composed of an ion-conducting portion (F₀) and a catalytic portion (F₁). The subunit composition of F₁ is ₃₃. The active ₃₃ oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F₁ (TF₁). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F₀. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of ₃₃. The torque of the rotation is estimated to be 420 pN·å from the H⁺ and H⁺-current through F₀F₁. The angular velocity () of is the rate-limiting step, because H⁺ increased theV _max of H⁺ current through F₀, but not theK _m (ATP). The rotational unit of F₀ (=ab₂c₁₀) is /5, while that in ₃₃ is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN₃-ATP), and tight Mg-ATP binding in ₃₃ was shown by ESR. The rotational relaxation of TF₁ is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of ₃₃ measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF₁) and F₀-d to hold free rotation of ₃₃.     

Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy

An NMR approach for determining the electron self-exchange (ESE) rate constants in blue copper proteins is presented. The approach uses the paramagnetic relaxation enhancement of resonances in 1D ¹H super-WEFT spectra of partly oxidized (paramagnetic) proteins. These spectra allow a more precise determination of the relevant paramagnetic linebroadenings than conventional 1D ¹H spectra and, thus, permit a more detailed investigation of the applicability of the linebroadenings for determining the electron exchange rates. The approach was used to estimate the ESE rate constant of plastocyanin from Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, unless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complication can be overcome by a correct analysis of the paramagnetic broadening of the combined double signals. Because of the high resolution and specific sensitivity of the approach it should be generally applicable to estimate electron transfer rates, k, if the paramagnetic relaxation enhancement R _2p of the resonances can be determined, and the conditions kR _2p or _pkR _2p are fulfilled, _p being the frequency separation between corresponding diamagnetic and paramagnetic sites.     

Unusual amino acids VIII. Asymmetric hydrogenation of some heteroaryl-N-CBZ and N-BOC aminocinnamic acid derivatives

Summary (Z)--[(Benzyloxy)- or (tert.-butyloxy)carbonylamino]- (thienyl)-or (furyl)-acrylic acids and their esters were prepared by known methods and hydrogenated to the corresponding optically active alanine derivatives with optical yields in the range of 58–93% ee using the cationic rhodium complex of PROPRAPHOS.     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.     

Hydrogen isotope discrimination in higher plants: Correlations with photosynthetic pathway and environment

Summary The ratio of deuterium to hydrogen (expressed as D) in hydrogen released as water during the combustion of dried plant material was examined. The D value (metabolic hydrogen) determined on plant materials grown under controlled conditions is correlated with pathways of photosynthetic carbon metabolism. C₃ plants show mean D values of-132 for shoots and -117 for roots; C₄ plants show mean D values of -91 for shoots and-77 for roots and CAM plants a D value of-75 for roots and shoots. The difference between the D value of shoot material from C₃ and C₄ plants was confirmed in species growing under a range of glasshouse conditions. This difference in D value between C₃ and C₄ species does not appear to be due to differences in the D value (tissue water) in the plants as a result of physical fractionation of hydrogen isotopes during transpiration. In C₃ and C₄ plants the hydrogen isotope discrimination is in the same direction as the carbon isotope discrimination and factors contributing to the difference in D values are discussed. In CAM plants grown in the laboratory or collected from the field D values range from-75 to +50 and are correlated with ¹³C values. When deprived of water, the D value (metabolic hydrogen) in both soluble and insoluble material in leaves of Kalanchoe daigremontiana Hamet et Perr., becomes less negative. These changes may reflect the deuterium enrichment of tissue water during transpiration, or in field conditions, may reflect the different D value of available water in areas of increasing aridity. Whatever the origin of the variable D value in CAM plants, this parameter may be a useful index of the water relations of these plants under natural conditions.     

Long Range Force between Pre-Replication Complexes (Pre-RC) in DNA Controls Replication and Cell Cycle Progression

A nonstationary interaction, that controls DNA replication and the cell cycle, is derived from a manybody physics model in a chemically open T cell. The model predicts a long range force F()=-(/2) (1-)(2-) between the pre-replication complexes (pre-RCs) bound by DNA, =/N being the relative displacement of preRCs, the number of pre-RCs, N the threshold for initiation, and the compressibility modulus in thelattice of pre-RCs which behaves like an elastically braced string. Initiation of DNA replication is induced by a switch of sign of F(), from attraction (-)and assembly in the G ₁ phase (0 < < N), to repulsion (+) and partialdisassembly in the S phase (N < < 2N), with release of licensing factors from the pre-RCs, thus explaining prevention of re-replication. Replication is terminated by a switch of sign of F at = 2N, when all primed replicons are duplicated once, and F(0)=0 corresponds to a resting cell in absence of driving force at = 0. The switch of sign of force at = N also explains the dynamic instability in growing microtubules (MTs), as well as switch in the interleukin-2 (IL2) interaction with its receptor in late G ₁, at the restriction point. Shape, slope and scale of the response curves derived agree well with experimental data from dividing T cells and polymerizing MTs, the variable length of which is due to anonlinear dependence of the growth amplitude on the initial concentrations of tubulin dimers and guanosine-tri-phosphate (GTP).     

The role of intracellular zinc in modulation of life and death of Hep-2 cells

Varying intracellular concentrations of zinc in laryngeal Hep-2 cells in relation to changing cultivation conditions in vitro were determined by atomic absorption spectrophotometry. Upon standard cultivation in DMEM with 10% serum, the mean concentration of zinc was determined at 0.88 ± 0.09 g/mg protein, with substantially decreased values in the cells exposed to a low-serum medium. Next, the study of the effects of a series of physiological and supraphysiological concentrations of ZnSO₄ on laryngeal cells and their correlation with determined intracellular concentrations of zinc was performed. It was found that zinc concentrations above 100 M were toxic to Hep-2 cells, inducing cell death in the interval of 96 h as determined by videomicroscopy, selective nuclear staining, and immunofluorescence detection of caspase-3 and specific cytokeratin 18 fragment. Both types of cell death were observed, with apoptosis being induced at moderately toxic zinc concentration of 150 M and necrosis at higher zinc concentrations of 300 M and 750 M, respectively. Lower concentrations (1.5–100 M), on the other hand, did not produce any measurable changes in cell morphology and function in the same time interval. Zinc at concentration of 1.5 M was found to slightly enhance proliferation of Hep-2 cells up to the certain time point, which seemed to correlate with maximal tolerable momentary intracellular level of zinc. These results illustrate the importance of determining the intracellular levels of zinc when trying to characterize the effect of exogenous zinc on life and death of laryngeal cells.     

Structural comparisons of serologically identical IA- and IE-encoded antigens from inbred and wild mice

The IA and IE products of B10.S(9R), B10.A, B10.KPB128, and B10.GAA37 were analyzed for primary structural variations by comparative tryptic peptide mapping. The A,A , andE products of B10.S(9R) and B10.A differed in about 40% of their acid-soluble tryptic peptides, indicating that intra-I-region recombinant strain B10.S(9R) received the genes encoding A, A, and E from theH- 2 ^s parental chromosome rather than fromH- 2 ^a . The tryptic peptides of E chains from B10.S(9R) and B10.A were indistinguishable, suggesting that B10.S(9R) received the gene encoding the E chain from theH- 2 ^a parental chromosome. Consistent with the results of others, these data suggest that the genes encodingA ,A and E chains are centromeric to theIJ subregion, while the gene encoding E chains is telomeric toIJ. The I-region products of two congenic lines carrying wild-derivedH- 2 haplotypes on a C57BL/10 background, designated B10.KPB128 and B10.GAA37, are serologically indistinguishable from those of B10.S(9R). The IA and IE products of B10.S(9R) were compared with those of B10.GAA37 and B10.KPB128 to determine the structural similarity of serologically identical products from allopatric populations of wild mice. The A,A , and E products of B10.S(9R) were indistinguishable from those of B10.GAA37 and B10.KPB128 by comparative tryptic peptide mapping. The E chains of these three lines differed in one or two of their acid-soluble tryptic peptides. The results indicate that the IA-encoded products of these three lines are structurally very similar and may be identical suggesting that some alleles of the A, A, and E chains may be maintained in stable linkage associations in allopatric populations of wild mice. The minor structural variations detected in the E chains of these three congenic lines indicate that the E chain is encoded by chromosome 17 and suggest that allelic E chains exhibit considerably less structural variability than other I-region encoded antigens.     

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work     

Fluorescence studies of normal and sickle beta apohemoglobin self-association

The acrylamide quenching of the intrinsic tryptophanyl fluorescence of normal and sickle apohemoglobins has been studied in 0.05 M potassium phosphate buffer,pH 7.5, at 5°C over a protein concentration range from 1 to 50M. Analysis of quenching dynamics revealed a strong dependence on acrylamide concentration for the intrinsic fluorescence of both normal and sickle apohemoglobins, suggesting that one tryptophanyl residue [presumably that at position 37(C3)], was more accessible to collisional quencher than the other tryptophanyl residue [15(A12)]. Additional studies, which altered viscosity and subunit assembly experimental parameters, supported the assignment of residue 37 as the more dynamically accessible residue. Finally, the quenching data were also found to be dependent on protein concentration, implying that this difference in the mobility between the two residues is a sensitive probe of self-aggregation. Extrapolated dynamic quenching constants at low concentration of acrylamide were used to estimate the dimer-monomer equilibrium dissociation constants of normal and sickle apohemoglobins, and were found to be 5.6 and 2.4M, respectively, thus demonstrating distinct self-association properties of _A and _S apohemoglobins.     

Immunocytological detection and localization of a peptide reacting with an α-endorphin antiserum in the corticotropic and melanotropic cells of the trout pituitary (Salmo irideus Gibb)

Summary In the pituitary of the trout, the corticotropic and melanotropic cells display a strong immunocytological reaction with -endorphin antiserum. This reaction persists even when a-endorphin antisera treated with -1-24 ACTH or -MSH are used. In the absence of pharmacological tests on the endorphic potencies of the compounds involved in the immunoreaction, it is not yet clear whether this reaction is due to the presence of an -endorphin-like peptide or simply an immunologically related peptide without the properties of endorphin. However, the presence of such peptides in the fish pituitary is interesting from the comparative point of view.     

Arabidopsis ammonium transporters,AtAMT1;1 and AtAMT1;2, have different biochemical properties and functional roles

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (K_m for ammonium and methylammonium of 8 M and 31 M, respectively) matched those of AtAMT1;1 expressed in yeast (K_m for methylammonium of 32 M and K_i for ammonium of 1–10 M). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (K_m for ammonium of 46 M). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a K_m of 36 M, a low-affinity phase with a K_m for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.     

Anti-diabetic activity of <Emphasis Type="Italic">β</Emphasis>-glucans and their enzymatically hydrolyzed oligosaccharides from <Emphasis Type="Italic">Agaricus blazei</Emphasis>

-Glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of -glucans were 30–50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing -glucans with an endo--(16)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though -glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of -glucans with respect to anti-diabetic activity.     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.