Novel drug delivery system to bone using acidic oligopeptide: pharmacokinetic characteristics and pharmacological potential.

We synthesized fifteen oligopeptides consisting of Asp or Glu conjugated with a fluorescent probe, 9- fluorenylmethylchloroformate (Fmoc). In the in vitro binding assay to putative hydroxyapatite (HA), the affinities of these conjugates depended only on the number of amino acid residues, not on their optical characters (L or D) or their species (Asp or Glu). In an in vivo experiment involving a single i.v. injection of Fmoc-D-Asp oligopeptides into mice, peptides consisting of over six Asp residues were selectively distributed to the bone. Then, we synthesized estradiol-17 beta-succinate-(L-Asp)6 [E2-(L-Asp)6] and studied its pharmacokinetic characteristics and its antiosteoporotic effects on ovariectomized (OVX) mice. Although the distribution volume of E2-(L-Asp)6 was significantly smaller than that of E2, E2-(L-Asp)6 was selectively distributed in the bone after i.v. injection and gradually decreased during 7 days. E2-(L-Asp)6 effectively prevented OVX-induced bone loss, without altering the uterine weight, in the dosage range of 0.11 to 1.1 mumol/kg once a week, while E2 increased both the bone mineral density and uterine weight at 0.37 mumol/kg every third day. The results suggest that acidic oligopeptide may be useful for drug delivery to bone and E2-(L-Asp)6 is a good candidate as an anti-osteoporosis drug without the adverse side effects of E2.

Myb-binding site regulates the expression of glucosamine-6-phosphate isomerase in Dictyostelium discoideum.

A homolog of the glucosamine-6-phosphate isomerase in the cellular slime mold Dictyostelium discoideum has been analyzed. The gene disruption mutant was arrested at the mound stage, demonstrating that the gene is important for development. The gene was expressed in vegetatively growing cells, silenced on starvation and expressed again in prestalk cells during the multicellular stages. The upstream region of the gene (1376 bp relative to ATG) was cloned and sequenced to study the transcription control mechanisms. Analysis of deletion mutants and a site-directed mutant indicated that the Myb-binding sequence (5'-AACTG-3') localized in the upstream region is important for gene expression. The results of gel-shift assays showed the presence of an Myb-related protein binding to the sequence at the growing phase and another protein binding to the sequence at developmental stages.

Interaction of phytoestrogens with estrogen receptors alpha and beta.

The human estrogen receptor (hER) exists as two subtypes, hER alpha and hER beta, that differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activities of soy isoflavones after digestion with enteric bacteria in competition binding assays with hER alpha or hER beta protein, and in a gene expression assay using a yeast system. The estrogenic activities of these isoflavones were also investigated by the growth of MCF-7 breast cancer cells. Isoflavone glycoside binds weakly to both receptors and estrogen receptor-dependent transcriptional expression is poor. The aglycones bind more strongly to hER beta than to hER alpha. The binding affinities of genistein, dihydrogenistein and equol are comparable to the binding affinity of 17 beta-estradiol. Equol induces transcription most strongly with hER alpha and hER beta. The concentration required for maximal gene expression is much higher than expected from the binding affinities of the compounds, and the maximal activity induced by these compounds is about half the activity of 17 beta-estradiol. Although genistin binds more weakly to the receptors and induces transcription less than does genistein, it stimulates the growth of MCF-7 cells more strongly than does genistein.

Complete sequences and expression kinetics of racG, racH, racI and racJ genes in Dictyostelium discoideum.

We sequenced and characterized the expression patterns of the genes (racG, racH, racI and racJ) in the Rho-family. The nucleotide sequences of these genes suggest that racI would be a pseudogene, while the other genes are likely to encode typical Rac proteins which contain either GTP-binding domain or CAAX prenylation motif as observed in other members of the family. The Northern blot analyses show that the expression patterns of these genes are distinctively regulated during development. The racG gene is expressed at almost the same level from the vegetative to the slug stage, but the amount of its transcript gradually decreases after culmination. Expression of the racJ gene is undetectable at the vegetative stage, becomes observable at the mound stage, reaches a peak at the slug stage and then suddenly disappears in the culmination stage. The racH gene is expressed in two forms of transcripts, both of which are undetectable at the vegetatively growing stage but abruptly increase in amount after starvation. Southern blot hybridization analysis demonstrates that these transcripts were derived from a single copy of the gene. Such distinct kinetics of the expression patterns suggests that these genes would have unique roles in Dictyostelium development.

Selective delivery of estradiol to bone by aspartic acid oligopeptide and its effects on ovariectomized mice.

We have developed a novel osteotropic prodrug of estradiol (E(2)) conjugated with L-Asp-hexapeptide (E(2).3D(6)), which has very low affinity for estrogen receptors, and in this study, we examined its pharmacokinetic behavior and pharmacological potential. After a single iv injection of E(2) x 3D(6) to mice, the half-time for elimination from plasma was about 100 min; however, E(2) was selectively delivered to the bone and eliminated very slowly, declining to the endogenous level at about 7 days. After a single iv injection of E(2), the half-time in plasma was about 70 min, whereas E(2) was highly distributed to the uterus, and the bone concentration of E(2) was only slightly increased at 6 h. When E(2) (0.37 micromol/kg, sc, every third day) or E(2) x 3D(6) (0.11 to 1.1 micromol/kg, sc, every seventh day) was administered to OVX mice for 4 weeks, E(2) increased the bone mineral density (BMD) together with weights of liver and uterus, whereas E(2) x 3D(6) increased only the BMD, in a dose-dependent manner. E(2) x 3D(6) enhanced the expression of messenger RNAs of bone matrix proteins (osteopontin, bone sialoprotein, type I collagen alpha) of OVX mice at 4 h after administration, but E(2) did very slightly. These results indicate that the E(2) prodrug was delivered to the bone, where it gradually released E(2), thereby ameliorating bone loss. This acidic oligopeptide appears to be a good candidate for selective drug delivery to bone.

Characterization of adriamycin-induced G2 arrest and its abrogation by caffeine in FL-amnion cells with or without p53.

We investigated the effect of Adriamycin on FL-amnion (FL) cells. After treatment with the drug, the cells arrested at G2, but we did not detect an increase in the p21 levels. We established a p53-deficient derivative of these cells, in which G2 arrest also occurred after treatment with Adriamycin, suggesting that the arrest we observed in these cells is independent of the p53 pathway. Low doses of Adriamycin (100-200 ng/ml) induced G2 arrest, while late S-phase arrest was observed at high doses (500-1000 ng/ml) in both FL and p53-deficient FL cells. Accumulation of cyclin B1 was detected only in cells arrested at G2, and not in those arrested at S phase, suggesting that the S-phase checkpoint functioned efficiently even in p53-deficient FL cells. In both cell lines, caffeine-induced activation of CDC2 kinase was detected only in cells arrested at G2 and CDC2 kinase-activated cells died exhibiting features of apoptosis. CDC2 kinase activation was inhibited by cycloheximide. Furthermore, cycloheximide inhibited activation of CDK2:cyclin A, which normally precedes CDC2 kinase activation in caffeine-treated cells. These results suggest that p53 and p21 do not have special roles in the S- and G2-phase checkpoints and that CDK2:cyclin A could be the target of the G2-phase DNA damage checkpoint.

Analysis of the disruption mutant of the oscillin homolog gene of Dictyostelium discoideum.

A homolog of oscillin, the Ca2+ oscillation-inducing factor of the hamster, was identified from the cellular slime mold Dictyostelium discoideum and designated Dd-oscillin. In the developmental stages of D. discoideum, the gene is expressed at the prestalk region which contains a higher concentration of cytosolic Ca2+ than the prespore region. The Dd-oscillin null strain aggregated but did not develop further when the cells were plated on non-nutrient agar at a density of 1.5x10(6) cells/cm2, showing that the Dd-oscillin gene is important for development. Since the null cells carrying the hamster oscillin gene formed fruiting body, the hamster oscillin was the homolog of Dd-oscillin as far as function is concerned. In addition, the null cells formed fruiting body in the presence of 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ: a specific inhibitor of Ca2+-ATPase activity in the endoplasmic reticulum). These results suggest that Dd-oscillin will increase cytosolic Ca2+ in the cells and promote further development.

Multiple centrosome formation induced by the expression of Vpr gene of human immunodeficiency virus.

We previously established a cell line called MIT-23 in which expression of the Vpr gene of human immunodeficiency virus 1 (HIV-1) can be controlled by the addition of tetracycline. Vpr expression induces multiple nuclear formation and increased ploidy in MIT-23 cells. We herein report that multipolar mitotic spindles were formed upon induction of Vpr. Further analysis of centrosomes with anti-gamma-tubulin immunostaining revealed that a significant population of cells 1 week after expression of Vpr gene product had an increased number of centrosomes in the cells with abnormal nuclei. Taking into account that the centrosome plays an important role in genome integrity, the abnormal number of centrosomes in cells expressing Vpr may be directly related to aneuploidy or the formation of micronuclei in MIT-23 cells, suggesting that Vpr has an oncogenic role in HIV infected cells.

Identification of the region required for monomerization of the rolling circle plasmid pKYM.

Plasmid pKYM, isolated from the gram-negative bacterium Shigella sonnei, is a small multicopy plasmid which replicates by a rolling circle mechanism. The formation of multimers has been observed in a derivative of pKYM which lost a part of the origin region, and the loss of the monomerization mechanism would have led to these multimers. By analyzing the constructs of several mutants, we discovered that a DNA region required for monomerization was present upstream of the RepK binding site in the replication origin. As either of the T-rich sequence or the inverted repeat sequences which were seen in that region have been lost in the multimer-forming plasmids, these sequences may be necessary for monomerization.

Rolling-circle plasmid pKYM re-initiates DNA replication.

It is believed that rolling-circle plasmids are incapable of re-initiation since they have to maintain their copy number and this is one of the differences between plasmids and phages as phi-x174. To examine whether a rolling-circle plasmid pKYM is incapable of re-initiating DNA replication, we constructed a plasmid that carries both the pKYM origin (fragment 13, 173 bp) and its truncated origin (fragment 32, 56 bp) in the same orientation. This plasmid yielded two smaller plasmids in the presence of RepK, an initiator protein. We showed that RepK can bind to the fragment 13 but not to fragment 32 which lacks the 3'- moiety of fragment 13. These results imply that RepK initiates DNA replication from fragment 13 and terminates at fragment 32, then the same RepK is used for re-initiation of replication from the fragment 32 region. pKYM is likely to be a unique plasmid that re-initiates DNA replication like a phase phi-x174.

Synthesis of RepK of rolling circle plasmid pKYM is regulated by countertranscript and HU protein.

Replication of rolling circle plasmid pKYM was regulated by RepK, a plasmid-encoded initiator protein, with HU protein and antisense RNA (copRNA) that block the expression of RepK. HU protein bound to the repK promoter in the presence of RepK protein and inhibited the transcription of repK mRNA. The copRNA would hybridize to repK mRNA and induce a stem-loop structure in which the repK Shine-Dalgarno sequence is sequestered by base pairing. Sequence substitution experiments demonstrated that this stem-loop not only inhibits translation but induces premature termination.

HU protein binding to the replication origin of the rolling-circle plasmid pKYM enhances DNA replication.

The RepK protein, which is encoded by the rolling-circle plasmid pKYM, binds to the PR I site in the pKYM DNA replication origin. We have identified HU as a protein that binds to the PR II and PR III sites in the replication-enhancing region which is downstream of PR I. DNA footprinting assays show that HU binds to these two sites only when RepK is bound to PR I, and that HU also enhances the binding of RepK to PR I. In vivo, pKYM was unable to transform an HU null strain. Two mutant RepK proteins, RepKW179Y, which contains a Trp-to-Tyr exchange at position 179, and RepKD277L, which contains an Asp-to-Leu mutation at residue 277, initiate DNA replication in vivo in the absence of HU. In vitro, these mutant RepK proteins form more stable complexes with the pKYM origin region than does the wild-type RepK protein. These results indicate that HU plays a role in the formation of a stable RepK-origin complex, which is required for the initiation of pKYM DNA replication.

Characterization of the genes encoding integrative and excisive functions of Lactobacillus phage øg1e: cloning, sequence analysis, and expression in Escherichia coli.

øg1e is a temperate phage of the Lactobacillus strain G1e. The phage-host junctions attR and attL cloned from the lysogen have a 24-bp common (core) sequence implicated in recombination. DNA sequencing analysis of a 5.2-kbp SacI fragment of the øg1e phage genome (42.5 kbp) revealed two possible open reading frames (ORF), xis and int, and the phage attachment (recombination) site (attP), whose 24-bp sequence is identical to the core sequence detected in attR and attL. The deduced int product (Int) is a basic protein of 391 amino acids with an estimated pI of 9.70, and significantly resembles other presumed integrases encoded by the Lactobacillus and Lactococcus phages including øadh and øLC3, as well as the Escherichia coli phages such as lambda. The predicted øg1e xis protein (Xis) is small and very acidic (66 amino acids; pI 4.55), and shows a resemblance (32% overall identity) with a putative excisionase encoded by the Staphylococcus phage ø11. The øg1e Int with a deduced molecular mass of 45.5 kDa was overproduced in E. coli cells, and electrophoretically analyzed.

Purification and DNA-binding properties of the integrase protein Int encoded by Lactobacillus plantarum phage.

The Lactobacillus plantarum temperate phage phi g1e (42,259 bp) encodes an integrase gene int linked to a phage attachment site attP (Kakikawa et al., 1997). To investigate phi g1e recombination, the integrase protein Int was overproduced in Escherichia coli under the T7 promoter, and purified. The Int protein had an apparent molecular mass of 42.0 kDa, corresponding well with that (45.5 kDa) predicted from the DNA sequence. Amino-acid sequencing revealed that the N-terminal 20 amino-acids of the purified Int protein completely coincided with those deduced from the DNA sequence, although deficient in the first methionine. Gel mobility-shift assays demonstrated that Int bound specifically to the attP region. In addition, footprinting analysis showed that Int protected about 35 bases, containing the 24-bp core domain at attP, from DNase I attack. These results are indicative of site-specific interaction of Int with the attP site, the reaction prerequisite for integration and excision of the phi g1e genome into and/or out of the host chromosome.

The role of Sertoli cells in the differentiation and exclusion of rat testicular germ cells in primary culture.

We have previously established a primary co-culture of spermatogenic and somatic cells of the rat testis, in which spermatogenic cells differentiate to some extent in terms of the occurrence of testis-specific gene expression. In the present study, the interaction between spermatogenic and somatic Sertoli cells was investigated in this culture system. Spermatogenic differentiation did not proceed when these two cell types were placed on opposite sides of a permeable membrane, thus avoiding direct contact. Further, a significant proportion of spermatogenic cells died by apoptosis during culture, and Sertoli cells engulfed and digested the degenerating spermatogenic cells. These results indicate that Sertoli cells participate both in the differentiation of spermatogenic cells and in the exclusion of degenerating spermatogenic cells, by directly attaching to those cells.

Determination of the single strand origin of Shigella sonnei plasmid pKYM.

The Shigella sonnei plasmid pKYM replicates by a rolling-circle mechanism in Escherichia coli. A 571 nucleotides HincII restriction fragment of the pKYM DNA harbors two potential hairpin loops (I and II). We cloned the fragment into a -ori defective M13 vector phage, M13 delta lac183. The chimera phage, MDKY5, showed a larger plaque size, and increased phage yield and rate of progeny replicative form DNA (RF) synthesis. Rifampicin reduced rate of conversion of the single- to double-stranded RF DNA. In addition, we introduced nucleotide deletions within the cloned pKYM DNA, by Bal31 nuclease digestion. Each of the deletion mutants thus constructed was lacking in a sequence containing the hairpin loops and formed smaller plaques. The in vivo analyses revealed that a 136 nucleotides sequence containing the two hairpins I and II is the pKYM minus origin for complementary strand synthesis (single strand origin, referred to as SSO) and harbors a recognition site(s) by host E. coli RNA polymerase, for primer RNA synthesis. Moreover, we found a 24 nt sequence, upstream of the SSO domain having 83% homology to the recombination site A (RSA) which functions in plasmid sitespecific recombination and/or transfer.

Transcription stimulation of the adenovirus type 12 E1a gene in vitro by the 266-amino-acid E1A protein.

We previously showed that the 13S but not the 12S mRNA product of the E1a gene of the highly oncogenic type 12 adenovirus (Ad12) stimulates the expression of its own gene. In this study, the mechanism for the autoregulation of the Ad12 E1a gene was investigated in vitro. The 266-amino-acid E1A protein of Ad12 was synthesized in yeast cells and purified as a 57-kDa polypeptide. The purified Ad12 E1A protein stimulated transcription from the proximal promoter of its own gene but had almost no effect on that from the distal promoter. A 35-bp upstream region including a TATA box for the proximal promoter seemed to be sufficient for transcription stimulation by the E1A protein. The Ad12 E1A protein formed a complex with a TATA box-binding protein (TBP), as does the E1A protein of nononcogenic Ad serotypes. Moreover, the E1A protein significantly reduced the binding of TBP to a TATA sequence, while it did not affect the DNA-binding activity of nuclear factor I, a stimulatory protein of the distal transcription of the Ad12 E1a gene. These results suggest that the 13S mRNA product of the Ad12 E1a gene regulates the transcription of its own gene by modulating the activity of TBP.

The silencer of mouse Pgk-2 consists of two discrete DNA elements that individually have no effect.

Pgk-2 encodes a testis-specific phosphoglycerate kinase isozyme (PGK), and the expression of mouse Pgk-2 is activated during the spermatogenic pathway at the pachytene spermatocyte stage. We previously reported the identification of a silencer-like cis-acting element in a region between nucleotides (nt) -1404 and -685 of mouse Pgk-2, which could be responsible for the repression of Pgk-2 expression in somatic tissues and pre-meiotic testicular germ cells. In the present study, the silencer was precisely located within an 87-bp region between nt -882 and -796. This region contained two distinct sequences that individually bound factors present in nuclear extracts of mouse cultured cells and rat tissues. The two sequences, when aligned in tandem upstream from the Pgk-2 promoter, inhibited cat expression driven by the promoter in mouse erythroleukemia cells, whereas either sequence alone did not show any effect. The results indicate that the Pgk-2 silencer consists of two distinct DNA elements which do not individually influence promoter activity. Binding of distinct nuclear factors to each DNA element appeared to be required for the silencer action.

Phosphorylation of NF-I in vitro by cdc2 kinase.

Nuclear factor I (NF-I) was purified to near homogeneity from Ehrlich ascites tumor cells. Mouse NF-I consisted of two peptides with relative molecular masses of 53 and 55 kDa and bound to the authentic NF-I site of the adenovirus DNA with no significant affinity with the CCAAT box. The purified protein was efficiently phosphorylated in a reaction containing immunoprecipitates with an anti-cdc2 kinase antibody. This phosphorylation was abolished when a synthetic peptide containing the consensus phosphorylation site by cdc2 kinase was added in excess to the reaction. These findings indicate that mouse NF-I is phosphorylated in vitro by the cdc2 protein kinase.

A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila.

D-raf, a Drosophila homolog of Raf-1, plays key roles in multiple signal transduction pathways. Dsor1, a putative factor downstream of D-raf, was genetically identified by screening of dominant suppressors of D-raf. Dsor1Su1 mapped on X chromosome significantly suppressed the D-raf mutant phenotypes, and the loss-of-function mutations of Dsor1 showed phenotypes similar to those of the D-raf null mutations. Dsor1Su1 also significantly suppressed the mutations of other terminal class genes acting further upstream of D-raf. Molecular cloning of Dsor1 revealed its product with striking similarity to the microtubule-associated protein (MAP) kinase activator and yeast PBS2, STE7, and byr1. Our genetic results demonstrate the connection between raf and the highly conserved protein kinase cascade involving MAP kinase in vivo.