Determination of the Avogadro constant by counting the atoms in a 28Si crystal.

The Avogadro constant links the atomic and the macroscopic properties of matter. Since the molar Planck constant is well known via the measurement of the Rydberg constant, it is also closely related to the Planck constant. In addition, its accurate determination is of paramount importance for a definition of the kilogram in terms of a fundamental constant. We describe a new approach for its determination by counting the atoms in 1 kg single-crystal spheres, which are highly enriched with the 28Si isotope. It enabled isotope dilution mass spectroscopy to determine the molar mass of the silicon crystal with unprecedented accuracy. The value obtained, NA = 6.022,140,78(18) × 10(23) mol(-1), is the most accurate input datum for a new definition of the kilogram.

Cry j 1 isoforms derived from Cryptomeria japonica trees have different binding properties to monoclonal antibodies.

BACKGROUND: We identified five Cryptomeria japonica trees producing Cry j 1 isoforms that cannot be detected in a sandwich ELISA using two monoclonal antibodies, J1B01 and J1B07, suggesting that the binding affinity of these isoforms for both monoclonal antibodies is low. OBJECTIVES: The binding properties of the Cry j 1 isoforms produced by five trees to J1B07 and J1B01 were examined. The complementary DNA (cDNA) sequences of the Cry j 1 isoforms were also determined. METHODS: To clarify the binding properties of these Cry j 1 isoforms to J1B01 and J1B07, Cry j 1 was quantified in pollen samples collected from each of the five trees, by sandwich ELISAs using polyclonal antibodies and either J1B01 or J1B07. The cDNA sequences of isoforms with different binding properties were determined. To test the assumption that amino acid substitutions affect the binding affinities of Cry j 1 isoforms for monoclonal antibodies, cleaved amplified polymorphic sequences (CAPS) markers representing the putative polymorphisms were used to analyse additional trees. RESULTS: Four of the five trees produced Cry j 1 isoforms with extremely low binding affinity for J1B07, whereas the other tree produced two different isoforms with low binding affinity for either J1B01 or J1B07. Cry j 1-encoding cDNA sequences for one of the four trees and for the exceptional fifth tree indicate that amino acid substitutions at positions 55 and 352 in mature Cry j 1 affect its binding to J1B01 and J1B07, respectively. This was supported by the results of CAPS analysis. CONCLUSION: The existence of Cry j 1 isoforms with low binding affinity for either J1B01 or J1B07 was established. Furthermore, a single amino acid substitution is involved in this difference in binding affinity for each monoclonal antibody.

Identification of AFLP markers linked to a resistance gene against pine needle gall midge in Japanese black pine.

Bulked segregant and AFLP analyses of two mapping populations (R17 x S6 and R17 x S1) were used to identify markers linked to Rpgm, the only known gene responsible for resistance to pine needle gall midge in Pinus thunbergii Parl. Rpgm was found to be bracketed by ACCC/CCTTT(190) on one side at a distance of 6.6 cM and ACGT/CCCGC(250) at 15.3 cM on the other side. The segregation of these markers was analyzed in two other families in order to determine their phase and transferability. One of the two additional resistant parents carried ACCC/CCTTT(190) in the homozygous state while the marker was in coupling (plus marker allele linked with an R allele) in a resistant parent, R17. The marker ACGT/CCCGC(250) was in a repulsion phase in R17 and was not detected in the other two resistant pine trees. Out of four AFLP markers identified, only ACGT/CCAAT(290) was transferable in all resistant trees tested, although its phase was opposite for different trees. These results indicate that in applying those markers to select resistant trees, the phase state of the markers in each resistant tree with respect to Rpgm needs to be considered.

Differences in spatial autocorrelation between four sub-populations of Alnus trabeculosa Hand.-Mazz. (Betulaceae).

To investigate the spatial structure of Alnus trabeculosa Hand.-Mazz, we compared three sub-populations at Imaichi in Tochigi Prefecture and one sub-population at Juo in Ibaraki Prefecture, Japan. A total of 269 trees, covering 0.71 ha in total, were mapped and genetically analyzed using nine enzyme systems encoding 13 isozyme loci. There were no significant differences between the four sub-populations in terms of Na,Ne, H(o), H(e )and F(IS). However, according to spatial autocorrelation analysis, the Juo sub-population, which is younger than the others, showed an aggregation of multilocus genotypes, especially within 25 m radii. In contrast with the Juo sub-population, those in Imaichi showed no clear spatial structuring. In the three Imaichi sub-populations, gametic phase disequilibrium is attributable to heterogeneity of genotype frequencies, but in the Juo sub-population it seems to be due to other factors, connected with the composition of the trees in the sub-population and/or its founder population, gene flow and asexual propagation. To conserve as much as possible of the genes or genotypes in restricted areas, conservation of populations that do not show clear family structures, such as those in Imaichi, would be most effective.

Degradation of c-Fos protein expressed by N-methyl-D-aspartic acid in nuclear fractions of murine hippocampus.

In both nuclear and cytosolic fractions of murine hippocampus, constitutive expression was seen with Fra-2 protein, but not with other Fos family members tested including c-Fos, Fos-B and Fra-1 proteins. Fos-B protein was only detected in nuclear fractions. The systemic administration of N-methyl-D-aspartic acid (NMDA) induced marked and transient expression of c-Fos protein, but not other family members, in both hippocampal fractions 2 h later. In vitro incubation at 30 degrees C led to more rapid degradation of inducible c-Fos protein than constitutive Fra-2 protein in nuclear fractions obtained 2 h after the administration of NMDA, without significantly affecting that of both member proteins in cytosolic fractions. The addition of phosphatase inhibitors significantly delayed the initial degradation rate of inducible c-Fos protein, with concomitant facilitation of that of constitutive Fra-2 protein, in nuclear fractions. The addition of protease inhibitors also delayed the initial degradation of constitutive Fra-2 protein, without markedly altering that of inducible c-Fos protein, in nuclear fractions. Immunoprecipitation analysis revealed that NMDA induced phosphorylation of c-Fos protein on tyrosine residues in nuclear fractions to a lesser extent than that on serine residues 2 h after administration. These results suggest that NMDA signals may be propagated to the nucleus to induce both expression and degradation of c-Fos protein through a molecular mechanism associated with phosphorylation on serine and/or tyrosine residues in murine hippocampus.

Consolidation of transient ionotropic glutamate signals through nuclear transcription factors in the brain.

Long-lasting alterations of neuronal functions could involve mechanisms associated with consolidation of transient extracellular signals through modulation of de novo synthesis of particular functional proteins in the brain. In eukaryotes, protein de novo synthesis is mainly under the control at the level of gene transcription by transcription factors in the cell nucleus. Transcription factors are nuclear proteins with an ability to recognize particular core nucleotides at the upstream and/or downstream of target genes, and thereby to modulate the activity of RNA polymerase II that is responsible for the formation of mRNA from double stranded DNA. Gel retardation electrophoresis is widely employed for conventional detection of DNA binding activities of a variety of transcription factors with different protein motifs. Extracellular ionotropic glutamate (Glu) signals lead to rapid and selective potentiation of DNA binding of the nuclear transcription factor activator protein-1 (AP1) that is a homo- and heterodimeric complex between Jun and Fos family members, in addition to inducing expression of the corresponding proteins, in a manner unique to each Glu signal in murine hippocampus. Therefore, extracellular Glu signals may be differentially transduced into the nucleus to express AP1 with different assemblies between Jun and Fos family members, and thereby to modulate de novo synthesis of the individual target proteins at the level of gene transcription in the hippocampus. Such mechanisms may be operative on synaptic plasticity as well as delayed neuronal death through consolidation of alterations of a variety of cellular functions induced by transient extracellular signals in the brain.

Facilitated wound healing by activation of the Transglutaminase 1 gene.

Transglutaminase 1 (TGase 1) is a Ca(2+)-dependent enzyme which catalyzes epsilon-(gamma-glutamyl)lysine cross-linking of substrate proteins such as involucrin and loricrin to generate the cornified envelope at the cell periphery of the stratum corneum. We have shown that disruption of the TGase 1 gene in mice results in neonatal lethality, absence of the cornified envelope, and impaired skin barrier function. Based on the importance of TGase 1 in epidermal morphogenesis, we have now assessed its role in wound healing. In neonatal mouse skin, TGase 1 mRNA as well as keratin 6alpha was induced in the epidermis at the wound edges as early as 2 hours after injury and that expression continued in the migrating epidermis until completion of re-epithelialization. The TGase 1 enzyme co-localized on the plasma membrane of migrating keratinocytes with involucrin, but not with loricrin, which suggests the premature assembly of the cornified envelope. Similar injuries to TGase 1 knockout mouse skins grafted on athymic nude mice showed substantial delays in wound healing concomitant with sustained K6alpha mRNA induction. From these results, we suggest that activation of the TGase 1gene is essential for facilitated repair of skin injury.

Molecular mechanisms associated with long-term consolidation of the NMDA signals.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptors in the mammalian brain plays a central role in synaptic plasticity underlying refinement of neuronal connections during development, or processes like long-term potentiation (LTP), learning and memory. On the other hand, over-activation of glutamate receptors leading to neurodegeneration has been implicated in major areas of brain pathology. Any sustained effect of a transient NMDA receptor activation is likely to involve signaling to the nucleus and coordinated changes in gene expression. Classically, a set of immediate-early genes is induced first; some of them are themselves transcription factors that control expression of other target genes. This review deals with the induction of Fos, Jun and Egr (Krox) transcription factors in response to NMDA or non-NMDA (AMPA/kainate) ionotropic receptor agonists in vivo or in neuronal cultures in vitro. In addition, the mechanism of induction of a model immediate-early gene c-fos in response to Ca2+ influx through activated NMDA receptors or voltage-sensitive calcium channels is discussed. Both modes of calcium entry induce c-fos via activation of multiple signaling pathways that converge on constitutive transcription factors cAMP-response element-binding protein (CREB), serum response factor (SRF) and a ternary complex factor (TCF), such as Elk-1. In contrast to the traditional view of the NMDA receptor as a ligand-gated calcium channel, whose activation leads to calcium influx and activation of Ca2+/calmodulin-dependent kinases, recent evidence highlights involvement of the Ras/ mitogen-activated protein kinase (MAPK) pathway in the NMDA signaling to the nucleus.

PUVA-induced lichen planus pemphigoides.

A 72-year-old woman had suffered from parapsoriasis en plaque (large plaque type) controlled by topically applied psoralen ultraviolet A (PUVA) therapy. The parapsoriasis lesions gradually disappeared, but numerous tiny red papules with pruritus appeared over the forearms and lower legs 120 days after starting PUVA therapy. These papules developed to form violaceous plaques. Histological findings demonstrated the characteristics of lichen planus. Two months later, tense bullae developed on the plaques and on uninvolved skin of the limbs. These were subepidermal, with linear deposits of IgG and C3 along the basement membrane zone (BMZ) in immunofluorescence of peribullous skin, and immunodeposits of type IV collagen along the floor of the bullae. We therefore, diagnosed lichen planus pemphigoides (LPP). Using systemic and topical steroid therapy, the lesions rapidly resolved and there has been no recurrence. This case suggests that the combination of basal cell injuries caused by chronic inflammation and PUVA therapy could expose BMZ components to autoreactive lymphocytes and induce LPP.

Expression of APG-2 protein, a member of the heat shock protein 110 family, in developing rat brain.

APG-2 protein is a member of the heat shock protein 110 family, and it is thought to play an important role in the maintenance of neuronal functions under physiological and stress conditions. However, neither the tissue-distribution of APG-2 protein nor developmental change of its expression has been studied at the protein level. Therefore, we generated an antiserum against APG-2 protein and studied expression of this protein in rat brain and other tissues by use of the Western blot method. The results showed a high expression of APG-2 protein in various regions of the central nervous system (cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla pons, and spinal cord) throughout the entire postnatal stage. Similarly, a high level of APG-2 protein was detected in the whole brain of rat embryos and in adult rat tissues such as liver, lung, spleen, and kidney. In contrast, its expression in heart was high at postnatal days 1 and 3, but thereafter drastically decreased to a low level. Furthermore, APG-2 protein was detected in neuronal primary cultures prepared from rat cerebral cortex, and its level did not change notably during neuronal differentiation. These results show that APG-2 protein is constitutively expressed in various tissues and also in neuronal cells throughout the entire embryonic and postnatal period. suggesting that it might play an important role in these tissues under non-stress conditions.

Gene transcription through Myc family members in eukaryotic cells.

c-Myc family proteins, encoded by c-myc family proto-oncogenes, play critical roles in mechanisms associated with proliferation, differentiation and apoptotic death in eukaryotic cells. These functions are mediated by transcriptional activity of these proteins through binding to the E-box core sequence CACGTG referred to as a Myc core element located at a promoter or enhancer region of the individual target genes in the nucleus. Recent studies have demonstrated the presence of novel nuclear proteins that specifically recognize a Myc core element, in addition to c-Myc, Max, Mad and Mxi1. On the other hand, a Myc core element has alternating purine/pyrimidine repeats which could undergo a conformational transition from right-handed (B-DNA) to left-handed (Z-DNA) forms in the presence of a high concentration of salts such as Mg2+ and polyamines. Similarly, a Myc element has a homopurine-homopyrimidine site that may take a triplex configuration in particular situations. We have searched for nuclear proteins that can specifically recognize a Myc core element in different topological variations in murine brain.

Predominant expression of nuclear activator protein-1 complex with DNA binding activity following systemic administration of N-methyl-D-aspartate in dentate granule cells of murine hippocampus.

The systemic administration of N-methyl-D-aspartate (100 mg/kg, i.p.) resulted in preferential but transient expression of the transcription factor activator protein-1 in the granule cell layers of the dentate gyrus in the murine hippocampus by maximally 700% 1 h later, without markedly affecting that in the pyramidal cell layers of the CA1 and CA3 subfields for 4 h. The potentiation was completely prevented by prior administration of the N-methyl-D-aspartate channel blocker dizocilpine at 10 mglkg. By contrast, kainate (40 mg/kg, i.p.) potentiated activator protein-1 DNA binding in adjacent areas around the pyramidal and granule cell layers, in addition to potentiating that in neuronal cell layers of the CA1 and CA3 subfields and the dentate gyrus. Light microscopic analysis revealed that kainate, but not N-methyl-D-aspartate, induced marked losses of the pyramidal cells in the CAI and CA3 subfields, without affecting the dentate granule cells, for 14 days after administration. Limited proteolysis by V8 protease and supershift, as well as immunoblotting assays using antibodies against c-Fos and c-Jun, invariably gave support for differential expression by N-methyl-D-aspartate and kainate of the activator protein-1 complex consisting of different partner proteins. Moreover, two-dimensional electrophoresis followed by immunoblotting analysis revealed the expression of several nuclear proteins immunoreactive with the anti-c-Fos antibody at molecular weights and isoelectric points clearly different from those of c-Fos itself in response to kainate, but not N-methyl-D-aspartate, in the hippocampus. These results suggest that in vivo N-methyl-D-aspartate signals are predominantly transduced into cell nuclei to express activator protein-1 complex through molecular mechanisms different from those for kainate signals in the granule cells of the dentate gyrus in the murine hippocampus.

Preventive effects of exogenous phospholipases on inhibition by ferrous ions of [3H]MK-801 binding in rat brain synaptic membranes.

Prior treatment with ferrous chloride led to marked inhibition of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of higher than 1 microM in rat brain synaptic membranes. Both phospholipases A2 and C significantly prevented the inhibition when treated before the treatment with ferrous chloride, while neither superoxide dismutase nor alpha-tocopherol affected the inhibition even when treated simultaneously with ferrous chloride. Of various saturated and unsaturated free fatty acids, moreover, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit binding when membranes were first treated with fatty acids, followed by the second treatment with ferrous chloride. These results suggest that membrane phospholipids may be at least in part responsible for interference by ferrous ions with opening processes of the native NMDA channel through molecular mechanisms associated with the liberation of unsaturated free fatty acids in rat brain.

Differential inhibition by ferrous ions of [3H]MK-801 binding to native N-methyl-D-aspartate channel in neonatal and adult rat brains.

In vitro addition or pretreatment with >/=1 microM ferrous chloride markedly inhibited in a concentration-dependent manner [3H]dizocilpine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in rat brain synaptic membranes. The addition of NMDA agonists invariably attenuated the inhibition of [3H]MK-801 binding in hippocampal synaptic membranes previously treated with ferrous chloride, without significantly affecting that in cerebellar synaptic membranes. In the absence of spermidine, ferrous chloride was more potent in inhibiting binding in the cerebral cortex and hippocampus in adult rats than in those in rats at 3 days after birth, while in the striatum [3H]MK-801 binding was 10 times more sensitive to inhibition by added ferrous chloride in neonatal rats than in adult rats. Addition of spermidine significantly attenuated the potency of ferrous chloride to inhibit binding in the cerebral cortex of adult rats, with facilitation of the inhibition in newborn rats. Moreover, spermidine significantly reduced the inhibitory potency of ferrous chloride in neonatal rat striatum, without markedly affecting that in adult rat striatum. These results suggest that ferrous ions may interfere with opening processes of the native NMDA channel through molecular mechanisms peculiar to neuronal development in a manner associated with the polyamine recognition domain.

Effect of ACE gene on diabetic nephropathy in NIDDM patients with insulin resistance.

We investigated the influence of the angiotensin-converting enzyme (ACE) gene on the onset and/or progression of diabetic nephropathy in 62 Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM; type II diabetes). Because a number of factors are believed to be involved in the onset and/or progression of diabetic nephropathy, especially in patients with NIDDM, we selected the patients with well-matched risk factors, duration of disease, glycemic control, blood pressure, and others. All patients had normal renal function and none were receiving ACE inhibitors. Patients were divided into three groups according to albumin excretion rate (AER): group A, patients with an AER less than 15 microg/min (n = 29); group B, patients with an AER between 15 and 70 microg/min (n = 19); and group C, patients with an AER greater than 70 microg/min (n = 14). The glucose disposal rate was estimated using a euglycemic hyperinsulinemic clamp. We determined the mean glucose disposal rate in 132 patients with NIDDM (6.49 mg/kg/min). Patients with a glucose disposal rate less than the mean rate were considered to have a high degree of insulin resistance (n = 36). The presence of an insertion/deletion (I/D) polymorphism of the ACE gene was determined by the polymerase chain reaction method. Among patients with a high degree of insulin resistance, diabetic nephropathy was present in 2 of 11 patients with the II genotype of the ACE gene compared with 19 of 25 patients with the ID or DD genotype (P = 0.0024). The prevalence of diabetic nephropathy was greater in patients with both significant insulin resistance and the D allele (19 of 25) than in the remaining patients (14 of 37; odds ratio, 5.20). These results suggest that the ACE gene influences the onset and/or progression of diabetic nephropathy in patients with NIDDM with significant insulin resistance.

Molecular weight distribution and the electrorheological response for anisotropic solutions of poly(gamma-benzyl-L-glutamate) induced by the convection of the electrohydrodynamic instability.

Transient responses of stress to a sinusoidal electric field were reported for anisotropic solutions of poly(gamma-benzyl-L-glutamate)s (PBLG's) with different distributions of the molecular weight. The transient stress, which was induced by the convection of the electrohydrodynamic instability, showed a steep and large increase when it was plotted against the instantaneous amplitude of the electric field. The maximum stress of a PBLG solution tended to increase with an increase in the maximum molecular weight of the PBLG in the molecular weight distribution.

Correlation between potentiation of AP1 DNA binding and expression of c-Fos in association with phosphorylation of CREB at serine133 in thalamus of gerbils with ischemia.

Protein biosynthesis is mainly under the control at the level of gene transcription in eukaryotes. Transcription factors are nuclear proteins with abilities to modulate the activity of RNA polymerase II which is responsible for the formation of messenger RNA from double stranded DNA in the cell nuclei. Binding of a radiolabeled oligonucleotide probe for the transcription factor activator protein-1 (AP1) was transiently potentiated 1 to 6 h after the recirculation of blood supply in the thalamus and striatum, but not in the entorhinal cortex, olfactory bulb, frontal cortex, cerebellar cortex and medulla-pons, in gerbils with transient global forebrain ischemia for 5 min, in addition to the hippocampal subregions. The ischemic insult not only increased the immunoreactivity with an antibody against cyclic AMP response element binding protein (CREB) phosphorylated at serine133, but also induced the expression of both c-Jun and c-Fos family proteins 3 h after the recirculation in the thalamus. Limited proteolysis by Staphylococcus aureus (S. aureus) V8 protease revealed the expression of different partner proteins of AP1 in response to ischemic signals in the thalamus. Moreover, ischemia for 2 min led to more prolonged elevation of AP1 binding in the thalamus at least up to 12 h after the reperfusion than that seen with ischemia for 5 min. These results suggest that potentiation of AP1 DNA binding may at least in part involve mechanisms associated with the expression of c-Fos protein through phosphorylation of CREB at serine133 in the thalamus of gerbils with ischemia.

[Calcium channels associated with glutamate receptors].

Ionotropic glutamate receptors are nowadays classified into two major categories based on the sensitivity to N-methyl-D-aspartic acid(NMDA) in the mammalian central nervous system. The NMDA channel has much higher permeability to Ca2+ than the non-NMDA channels in most situations, while point mutation could lead to increased permeation of Ca2+ across the non-NMDA channels. Opening of the NMDA channel is under control by a variety of endogenous low molecules, including glycine, polyamines, Mg2+, Zn2+ and H+, in addition to modulation by Ca2+/calmodulin, protein kinases and protein phosphatases. Moreover, the NMDA channel contains a redox site modulatory for the opening. Recent molecular cloning studies have revealed that the NMDA channel is a protein complex consisting of different subunits such as NMDAR1 and NMDAR2 with heterologous expression in the brain. Studies on mice knocked out of a particular gene argue in favor of an essential role of the corresponding subunits in molecular mechanisms underlying neuronal network formation and synaptic plasticity.

Possible involvement of activator protein-1 DNA binding in mechanisms underlying ischemic tolerance in the CA1 subfield of gerbil hippocampus.

Transcription factors are nuclear proteins with an ability to recognize particular nucleotide sequences on double stranded genomic DNAs and thereby modulate the activity of RNA polymerase II which is responsible for the formation of messenger RNAs in cell nuclei. Gel retardation electrophoresis revealed that transient forebrain ischemia for 5 min led to drastic potentiation of binding of a radiolabelled double-stranded oligonucleotide probe for the transcription factor activator protein-1, in the thalamus as well as the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of the gerbils previously given ischemia for 2 min two days before, which is known to induce tolerance to subsequent severe ischemia in the CA1 subfield. By contrast, ischemia for 5 min resulted in prolonged potentiation of activator protein-1 binding in the vulnerable CA1 subfield of the gerbils with prior ischemia for 5 min 14 days before, which is shown to induce delayed death of the pyramidal neurons exclusively in this subfield. Similar prolongation was seen with activator protein-1 binding in the vulnerable thalamus but not in the resistant CA3 subfield and dentate gyrus of the gerbils with such repeated ischemia for 5 min. Limited proteolysis by Staphylococcus aureus V8 protease as well as supershift assays using antibodies against c-Fos and c-Jun proteins demonstrated the possible difference in constructive partner proteins of activator protein-1 among nuclear extracts of the CA1 subfield obtained from gerbils with single, tolerated and repeated ischemia. These results suggest that de novo protein synthesis may underlie molecular mechanisms associated with acquisition of the ischemic tolerance through modulation at the level of gene transcription by activator protein-1 composed of different constructive partner proteins in the CA1 subfield. Possible participation of glial cells in the modulation is also suggested in particular situations.