Young's interference of electrons in field emission patterns.

We have observed interference fringes of electrons in field emission patterns from multiwalled carbon nanotubes at 60 K. The observed fringe pattern is reproduced by calculations based on the formula of Young's interference of two beams. Three-beam interference has also been detected over short time periods. We discuss the reason why Young's interference appears in the electron emission pattern in accelerating fields.

Differential regulation of liver-specific and ubiquitously-expressed genes in primary rat hepatocytes by the extracellular matrix.

Primary rat hepatocytes were cultured with an extracellular matrix (ECM) overlay, in order to investigate the effect of an ECM on gene expression in hepatocytes. When hepatocytes, isolated by the collagenase-perfusion method, were cultured on type I collagen-coated dishes, the mRNA levels of liver-specific genes (aldolase B, tyrosine aminotransferase and albumin) decreased continuously, while those of ubiquitously-expressed genes (glyceraldehyde 3-phosphate dehydrogenase and beta-actin) increased. When a dilute ECM derived from the Engelbreth-Holm-Swarm mouse sarcoma (an EHS gel) was added to the above hepatocytes 3 days after plating, the mRNA levels of liver-specific genes increased, while those of ubiquitously-expressed genes decreased. The effects of a rat liver biomatrix (a physiological ECM for rat hepatocytes) on gene expression in primary hepatocytes were similar to those of the EHS gel. A nuclear run-on assay, and 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole or actinomycin D treatments revealed that the transcriptional rates of liver-specific genes were enhanced by the EHS gel overlay, while the apparent stability of the corresponding mRNAs were unchanged. In contrast, the transcriptional rates of ubiquitously-expressed genes were not greatly affected by an EHS gel overlay, while the apparent stability of their mRNAs were decreased. These data suggest that the ECM plays an important role in the maintenance of the differentiated characteristics of primary hepatocytes by inducing the transcription of liver-specific genes and, also, by destabilizing the mRNAs of ubiquitously-expressed genes.

Characterization of the responsive elements to hormones in the rat aldolase B gene.

Transcription of the aldolase B gene, AldB, in the liver is regulated by hormones such as insulin and glucagon. To characterize the elements that are responsive to these hormones in the upstream region of AldB, plasmids carrying various length of the upstream region of this gene were constructed and transfected to primary cultured rat hepatocytes. The promoter activities were gradually increased by progressive deletion of the 5'-upstream region, and high activities were observed for constructs carrying the sequence between -408 and -85 bp, suggesting the presence of suppressive element(s) in the upstream region of -409 bp. The transcription activities of the mutants containing the sequences between -228 and -85 bp were enhanced by insulin, and glucagon suppressed the transcription activities of those containing the sequence between -764 and -85 bp. Two sequence elements similar to the cAMP-responsive element (CRE), one from -89 to -82 bp and another from +13 to +20 bp, were found in the upstream sequence of the gene. The latter element is not functional because its deletion did not affect either the transcription efficiency or glucagon response. However, the deletion of the former element diminished both functions. A gel retardation assay showed that the nuclear factor binds to the former element, which was competitive with authentic CRE oligonucleotide but not with the mutant CRE one. These results suggest that the CRE-like element in the promoter region is prerequisite for both fundamental transcription efficiency of the gene and suppression by glucagon in hepatocytes.

Change of liver function in hypertrophying lobe of rabbit liver after portal branch ligation.

Preoperative portal embolization (PE) is useful for the prevention of postoperative liver failure after extended hepatectomy. However, clinical evaluation of liver function in the hypertrophying lobe after PE has not been studied. Here we report functional changes in the hypertrophying lobe using a 80% portal-branch-ligation rabbit model. Liver function was evaluated by the expression of liver-specific genes detected by Northern blot analysis and plasma disappearance rate of indocyanine green (ICG). The weight of the unligated lobe after portal ligation increased about twofold on the 7th postoperative day (POD) and about threefold on the 14th POD. The mRNA levels of the liver-specific genes (albumin, aldolase B, and tyrosine aminotransferase) in the unligated lobe decreased to about 50% on the 1st POD and returned to the preoperative levels on the 7-14th POD. In contrast, the expression of histone H2B mRNA increased on the 3rd-7th POD. The plasma disappearance rate of ICG (K-ICG) in the rabbit that has only the unligated lobe did not significantly change during the first 7 days, but then improved and recovered to 80% of that in the rabbit that has whole liver on the 14th POD. These results indicate that liver function of the hypertrophying lobe after portal branch ligation does not increase during the first 7 days despite an increase in liver weight. This finding suggests that the compensatory hypertrophying liver is enlarging without functional augmentation in the early period after PE.

Expression of CD44 in human cumulus and mural granulosa cells of individual patients in in-vitro fertilization programmes.

CD44 is a polymorphic and polyfunctional transmembrane glycoprotein widely expressed in many types of cells. Here, the expression of this protein on human membrana granulosa was studied by two techniques. Using confocal laser scanning microscopy (CLSM) with the mouse monoclonal antibody to human CD44 (clone G44-26), cells immunoreactive for CD44 were observed in both cumulus and mural granulosa cell masses. On the other hand, using monoclonal antibody to human CD44v9, goat polyclonal antibody to human CD44v3-10 and the clone G44-26, no immunoreactivity for CD44v9 and/or CD44v3-10 was observed in either cell group by flow cytometry. In the flow cytometric analysis of 32 patients, the incidence of CD44 expression in cumulus cells (62.6+/-1.3%) was significantly higher than that in mural granulosa cells (38.5+/-3.2%) (P<0.0001). In the comparison of CD44 expression by flow cytometry according to the maturation of each cumulus-oocyte complex, the incidence of CD44 expression of cumulus cells was significantly higher in the mature group than in the immature group (P<0.05). In a flow cytometric analysis, patients with endometriosis showed a significantly lower incidence of CD44 expression in cumulus cells compared to the infertility of unknown origin group (P<0.05), and compared to both the male infertility group and the unknown origin group in mural granulosa cells (P<0.01). These findings suggest that the standard form of CD44 is expressed in human membrana granulosa with polarity and may play an important role in oocyte maturation.

Genistein induces p21(Cip1/WAF1) expression and blocks the G1 to S phase transition in mouse fibroblast and melanoma cells.

Genistein, the principal isoflavonoid in soybeans, is reported to inhibit cell cycle progression, but the molecular basis for this event is unknown. Here we show that genistein inhibits DNA synthesis and suppresses cyclin E-associated cyclin-dependent kinase-2 (CDK2) activity when quiescent BALB/c 3T3 fibroblasts are stimulated with serum. In these cells, a CDK2 inhibitor, p21(Cip1/WAF1), is markedly increased by genistein, but another CDK2 inhibitor, p27(Kip1), is not increased. In exponentially growing BALB/c 3T3 cells, genistein inhibits proliferation of the cells in a dose-dependent manner. Flow cytometric analysis and measurement of DNA synthesis indicate that genistein blocks the G1 to S phase transition of these cells, which is concomitant with G2-M arrest. In mouse B16-F1 melanoma cells, genistein also blocks the transition of G1 to S phase without arresting at G2-M at low doses. In both cell lines, genistein suppresses cyclin E/CDK2 activity and induces p21(Cip1/WAF1) expression. These results suggest that genistein affects the restriction point control of the cell cycle by inducing p21(Cip1/WAF1) expression in mouse fibroblast and melanoma cells.

Synergistic induction by collagen and fibronectin of liver-specific genes in rat primary cultured hepatocytes.

The extracellular matrix plays an important role for maintaining liver functions. We examined the effects of type I collagen and fibronectin on the expression of liver-specific genes in rat primary hepatocytes. When primary culture hepatocytes were overlaid with a type I collagen-gel, the expression of liver-specific genes (tyrosine aminotransferase, aldolase B, and albumin) increased by 4-5 times, compared with not overlaid hepatocytes. In contrast, the expression of non-liver-specific genes (GAPDH and beta-actin) was suppressed under the same conditions. The addition of fibronectin together with type I collagen-gel further enhanced the expression of liver-specific genes by 1.4-1.8 times. The addition of GRGDS peptide instead of fibronectin with the collagen-gel had a similar effect on hepatic gene expression to that of fibronectin. Addition of fibronectin alone exhibited had no effect on gene expression. These results suggest that type I collagen and fibronectin synergistically induce liver-specific genes.

Hormonal regulation of aldolase B gene expression in rat primary cultured hepatocytes.

Gene expression of aldolase B, an important enzyme for glucose and fructose metabolism, is regulated by hormones. We examined direct effects of major hormones on aldolase B gene expression in rat primary cultured hepatocytes, in comparison with those on the gene expression of phospho(enol)pyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis. Insulin, dexamethasone, and high concentration of glucose increased aldolase B mRNA abundance in the hepatocytes. Glucagon strongly suppressed aldolase B gene expression, and this hormone canceled the stimulative effects of insulin, dexamethasone, and high concentration of glucose. Epinephrine and thyroxine slightly reduced aldolase B mRNA abundance, but these hormones did not cancel the stimulative effects of insulin and dexamethasone. To the contrary, expression of PEPCK gene was suppressed by insulin, dexamethasone, and high concentration of glucose, and remarkably induced by glucagon. Glucagon rapidly suppressed aldolase B gene expression at the transcriptional level. Forskolin and dibutyryl cAMP mimicked the suppressive effect of glucagon on aldolase B gene expression. These results suggest that glucagon may be a key regulator of aldolase B gene transcription through a cAMP/protein kinase A-signaling pathway.

Loss of cell adhesion to substratum up-regulates p21Cip1/WAF1 expression in BALB/c 3T3 fibroblasts.

Cell adhesion to substratum is essential for the transition of G1 to S phase in mouse BALB/c 3T3 fibroblast cell cycle. Loss of cell adhesion in late G1 phase caused blockage of the G1/S phase transition and repression of cyclin E-associated cyclin-dependent kinase-2 (CDK2) activity. A CDK2 inhibitor abundant in quiescent cells, p27Kip1, was down-regulated by growth factors in serum, and this down-regulation was partially prevented by loss of cell adhesion. Another CDK2 inhibitor, p21Cip1/WAF1, which was undetectable in quiescent cells, was markedly induced by loss of cell adhesion. In exponentially growing cells, loss of cell adhesion also induced p21Cip1/WAF1 expression but did not affect the abundance of p27Kip1. These results suggest that loss of cell adhesion to substratum up-regulates p21Cip1/WAF1 expression, which plays an essential role for arresting the BALB/c 3T3 fibroblast cell cycle.

Functional localization of glucose transporter 2 in rat liver.

Heterogeneity of zonal hepatocytes is important to elicit specific liver function. We investigated the distribution of glucose transporter 2 (GLUT-2) in normal rat liver by immunostaining and Northern blot analysis. GLUT-2 stained by immunohistochemistry was distributed predominantly in the periportal hepatocytes and gradually thinned towards the perivenous zone. Ultrastructural immunostaining of GLUT-2 showed that it was localized on microvilli of the sinusoidal plasma membrane of hepatocytes but not on the basolateral plasma membrane. Consistent with the distribution of GLUT-2 protein, the level of GLUT-2 mRNA in periportal hepatocytes was 1.9-fold higher than in perivenous hepatocytes selectively isolated by the differential isolation technique. In addition, the mRNA level of phosphoenolpyruvate carboxykinase, one of the key enzymes of gluconeogenesis, was also twofold higher in the periportal hepatocytes. These results suggest that GLUT-2 contributes to the functional difference between periportal and perivenous hepatocytes in glucose metabolism of the liver.

Cell adhesion to substratum and activation of tyrosine kinases are essentially required for G1/S phase transition in BALB/c 3T3 fibroblasts.

Cell adhesion to substratum and activation of tyrosine kinases are essential for the progression of cell cycle through G1 phase in mammalian cells. The kinetic studies of mouse BALB/c 3T3 fibroblasts showed that serum was no longer required for the progression of G1/S phase transition. In contrast, cell adhesion was essentially required in late G1 phase, especially at the period of G1/S transition. Among the kinase inhibitors used to elucidate the signal transduction caused by cell adhesion, tyrosine kinase inhibitors, genistein and herbimycin A, blocked the G1/S transition most effectively when cells were exposed to the inhibitors at the period of G1/S transition. Cell adhesion was not critically required for cells to undergo DNA synthesis once they had passed the G1/S boundary, and the effects of tyrosine kinase inhibitors on the progression of S phase were also not critical. The expressions of histone H2B and dihydrofolate reductase (DHFR) genes (S phase specific genes) and also the transcription factor E2F-1 gene (an activator of DHFR gene) were suppressed when cells were cultured without adhesion or exposed to the tyrosine kinase inhibitors. These results suggest that cell adhesion to substratum plays an important role in the G1/S phase transition of mouse BALB/c 3T3 fibroblasts through the activation of tyrosine kinases other than growth factor receptor-tyrosine kinases.

Interleukin-6 down-regulates expressions of the aldolase B and albumin genes through a pathway involving the activation of tyrosine kinase.

Interleukin-6 plays a key role in mediating acute-phase protein synthesis in hepatocytes. However, the mechanism of how interleukin-6 regulates aldolase B and albumin syntheses in hepatocytes is not completely understood. In this study, using primary cultured rat hepatocytes, we have shown that interleukin-6 down-regulates expressions of the aldolase B and albumin genes in a dose- and time-dependent manner. We examined whether the decrease in aldolase B and albumin mRNA expressions by interleukin-6 reflected transcriptional down-regulation or stability of the mRNA. Actinomycin D and cycloheximide did not affect the interleukin-6-mediated decrease in the expressions of both genes. These results suggest that the decreased expressions of both genes induced by interleukin-6 is controlled at the transcriptional level, and that it is due neither to increased degradation of mRNA nor to synthesis of new proteins. Protein kinases play a fundamental role in the intracellular signal transduction. To examine the interleukin-6 signal pathway(s) leading to the decrease of aldolase B and albumin mRNA expressions, we tested various kinds of protein kinase inhibitors in this system. Herbimycin A, an inhibitor of tyrosine kinase(s), prevented the decrease in the expression of aldolase B and albumin mRNAs by interleukin-6. H-7, an inhibitor of protein kinase C, prevented the decrease in the expression of albumin mRNA by interleukin-6, but did not induce recovery of that of aldolase B mRNA. These results suggest that a tyrosine kinase(s) or a herbimycin A-sensitive kinase(s) constitutes a common pathway for interleukin-6-mediated reduction of aldolase B and albumin mRNA expressions and that distinct pathways exist for the modes of expression of the two mRNAs.

Dietary and hormonal regulation of aldolase B gene transcription in rat liver.

In the liver of the fasted rat, the aldolase B (AldB) mRNA level decreased to about half of that of the control rat. When the control rat was refed the glucose-rich diet, the AldB mRNA level increased about six to seven times more than in the fasted rat. This increase was shown as the activation of the AldB gene transcription by a nuclear run-on assay. To understand the causal factor(s) for this activation, the relationship between the AldB mRNA level in the liver and the plasma concentrations of hormones, which are known as major regulators of carbohydrate metabolism during fasting and refeeding, was investigated. The plasma insulin level in the rat which was refed the glucose-rich diet increased in parallel to AldB mRNA level, while the plasma glucagon level decreased reciprocally to it. The relationship of the plasma corticosterone level to the AldB mRNA level was not obvious. To directly confirm the effects of these hormones on AldB gene transcription in the liver, the responses of AldB gene in the primary cultured hepatocytes to these hormones were examined. Insulin and dexamethasone were effective to activate AldB gene, while glucagon and thyroxine were suppressive. Thyroxine did not extinguish the effects of insulin and dexamethasone, but glucagon canceled them. Thus, it is probable that in vivo these hormones synergistically regulate the AldB gene transcription. In vitro transcription analysis of two AldB promoter constructs suggested that the proximal half of the AldB promoter (up to -92 bp from the transcription start site) is, at least in part, involved for this induction, and the distal half which contains liver-specific elements (-93 to -202 bp) is not involved. The possible explanation for the dietary regulation of aldolase B gene transcription in the liver is discussed.

A novel growth-inducible gene that encodes a protein with a conserved cold-shock domain.

We have isolated a cDNA that encodes a novel member of the Y-box binding protein family, termed as RYB-a (Rat Y-box Binding protein-a). RYB-a is a 31 kDa protein that contains a conserved cold-shock domain and an amino acid alignment similar to those of charge zipper proteins. Expression of RYB-a mRNA was highly abundant in the skeletal muscle, spleen, and fetal liver. The expression is very low in new-born and adult livers, suggesting its expression is under developmental regulation. In addition, the expression of RYB-a mRNA was induced in the liver during regeneration and by stimulation of quiescent fibroblast cells with serum. Induction in the fibroblasts was inhibited by treating the cell with a specific tyrosine kinase inhibitor, genistein or by detachment of cell-adhesion. Since both treatments are known to inhibit G1 cells to enter S phase, RYB-a gene is thought to be a member of growth-inducible genes.

Eumelanin biosynthesis is regulated by coordinate expression of tyrosinase and tyrosinase-related protein-1 genes.

Melanin is specifically produced in melanocytes. The pathway for melanin biosynthesis is regulated by a number of melanocyte-specific proteins, including tyrosinase and tyrosinase-related protein-1 (TRP-1, b locus protein). To understand the regulation of melanogenesis, we examined tyrosinase activities, mRNA levels of tyrosinase and TRP-1, and eumelanin and pheomelanin contents in mouse B16-F1 melanoma cells after they had been treated with some melanotropic reagents. Cholera toxin, alpha-melanocyte-stimulating hormone, and dibutyryl cyclic AMP increased tyrosinase activity and stimulated eumelanin biosynthesis. These reagents elevated intracellular cAMP levels. In contrast, 12-O-tetradecanoylphorbol 13-acetate reduced tyrosinase activity and eumelanin synthesis. In all cases, the mRNA levels of tyrosinase and TRP-1 changed in parallel with tyrosinase activity and eumelanin content. TRP-1 was induced simultaneously with tyrosinase, although its inducibility was lower than that of tyrosinase. These results suggest that the expressions of tyrosinase and TRP-1 genes are coordinately regulated by melanotropic reagents through cAMP-dependent protein kinase and protein kinase C in mouse B16-F1 cells, and that their coordinate expression causes eumelanin biosynthesis.

Rat ribosomal protein L35a multigene family: molecular structure and characterization of three L35a-related pseudogenes.

The rat ribosomal protein L35a gene comprises a multigene family which contains 15-20 members as shown by the Southern blot analysis using L35a cDNA as a probe. We isolated 15 independent clones which contained distinct genes from a rat genomic library. Analysis of the restriction sites showed that all of them lacked the intervening sequences. Thermal stability of the hybrid molecules between these genes and the cDNA indicated that the similarity of the genes to the cDNA sequence varied. The nucleotide sequences of three genes gRL35a-A, gRL35a-B and gRL35a-G were determined. They shared some characteristics; namely: they lacked the intervening sequences, they contained (A)-rich tracts, and they were flanked by direct repeats. Two genes, gRL35a-A and gRL35a-B, contained a sequence completely identical to that of the cDNA. The nucleotide sequence of the 5' flanking region of gRL35a-B showed a significant homology with that of the same region of mouse ribosomal protein L32-related unmutated processed genes. Although this region of gRL35a-B contained the sequences homologous to the TATA box and the CCAAT box, gRL35a-B was not transcribed in an in vitro assay system. Thus, the L35a gene family comprises mostly processed pseudogenes. Further, Southern blot analysis in various animals indicated that the multigene construction of this ribosomal protein gene was a feature of mammalian genes. The origin and the evolutionary aspect of processed pseudogenes are discussed.

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L31.

A cDNA clone, specific for rat ribosomal protein L31, was isolated by hybrid-selected translation from a cDNA library made for 8-9-S poly(A)-rich RNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. It consists of 25 base pairs from the 5' leading sequence, the entire coding sequence of 378 base pairs and the 3' trailing sequence of 36 base pairs besides the poly(A) tail. The primary structure of protein L31 was deduced from the nucleotide sequence. It consists of 124 amino acids with a relative molecular mass of 14,331. The calculated amino acid composition is consistent with the reported composition determined for the hydrolysate of L31. The amino acid sequence showed marked homology with that of yeast ribosomal protein L34.

Immunological quantitation of tyrosinase from wild-type and albino mutant mice.

The relationship between gene dosage, enzyme activity, and level of immunologically cross-reacting material (CRM) was examined in mammalian tyrosinase (EC 1.14.18.1) by rocket immunoelectrophoresis. Skin extracts from mice heterozygous (C/c) and homozygous (c/c) for the albino locus contain 46% and 0% of CRM, respectively, as compared with wild-type (C/C) animals. Enzyme activity and CRM level were directly proportional in these genotypes, suggesting that the albino locus controls the quantity of tyrosinase produced in melanocytes.