Effect of HPMC and mannitol on drug release and bioadhesion behavior of buccal discs of buspirone hydrochloride: In-vitro and in-vivo pharmacokinetic studies.

Delivery of orally compromised therapeutic drug molecules to the systemic circulation via buccal route has gained a significant interest in recent past. Bioadhesive polymers play a major role in designing such buccal dosage forms, as they help in adhesion of designed delivery system to mucosal membrane and also prolong release of drug from delivery system. In the present study, HPMC (release retarding polymer) and mannitol (diluent and pore former) were used to prepare bioadhesive and controlled release buccal discs of buspirone hydrochloride (BS) by direct compression method. Compatibility of BS with various excipients used during the study was assessed using DSC and FTIR techniques. Effect of mannitol and HPMC on drug release and bioadhesive strength was studied using a 3(2) factorial design. The drug release rate from delivery system decreased with increasing levels of HPMC in formulations. However, bioadhesive strength of formulations increased with increasing proportion of HPMC in buccal discs. Increased levels of mannitol resulted in faster rate of drug release and rapid in vitro uptake of water due to the formation of channels in the matrix. Pharmacokinetic studies of designed bioadhesive buccal discs in rabbits demonstrated a 10-fold increase in bioavailability in comparison with oral bioavailability of buspirone reported.

Growth differentiation factor-15 encodes a novel microRNA 3189 that functions as a potent regulator of cell death.

According to the latest version of miRBase, approximately 30% of microRNAs (miRNAs) are unique to primates, but the physiological function of the vast majority remains unknown. In this study, we identified miR-3189 as a novel, p53-regulated, primate-specific miRNA embedded in the intron of the p53-target gene GDF15. Antagonizing miR-3189 increased proliferation and sensitized cells to DNA damage-induced apoptosis, suggesting a tumor suppressor function for endogenous miR-3189. Identification of genome-wide miR-3189 targets revealed that miR-3189 directly inhibits the expression of a large number of genes involved in cell cycle control and cell survival. In addition, miR-3189 downregulated the expression of multiple p53 inhibitors resulting in elevated p53 levels and upregulation of several p53 targets including p21 (CDKN1A), GADD45A and the miR-3189 host gene GDF15, suggesting miR-3189 auto-regulation. Surprisingly, miR-3189 overexpression in p53-/- cells upregulated a subset of p53-targets including GDF15, GADD45A, and NOXA, but not CDKN1A. Consistent with these results, overexpression of miR-3189 potently induced apoptosis and inhibited tumorigenicity in vivo in a p53-independent manner. Collectively, our study identified miR-3189 as a novel, primate-specific miRNA whose effects are mediated by both p53-dependent and p53-independent mechanisms. miR-3189 may, therefore, represent a novel tool that can be utilized therapeutically to induce a potent proapoptotic effect even in p53-deficient tumors.

Tissue- and development-specific induction and turnover of hsp70 transcripts from loci 87A and 87C after heat shock and during recovery in Drosophila melanogaster.

The haploid genome of Drosophila melanogaster normally carries at least five nearly identical copies of heat-shock-inducible hsp70 genes, two copies at the 87A7 and three copies at the 87C1 chromosome sites. We used in situ hybridization of the cDNA, which hybridizes with transcripts of all five hsp70 genes, and of two 3' untranslated region (3'UTR; specific for the 87A7- and 87C1-type hsp70 transcripts) riboprobes to cellular RNA to examine whether all these copies were similarly induced by heat shock in different cell types of D. melanogaster. Our results revealed remarkable differences not only in the heat-shock-inducibility of the hsp70 genes at the 87A7 and 87C1 loci, but also in their post-transcriptional metabolism, such as the stability of the transcripts and of their 3'UTRs in different cell types in developing embryos and in larval and adult tissues. Our results also revealed the constitutive presence of the heat-shock-inducible form of Hsp70 in a subset of late spermatogonial cells from the second-instar larval stage onwards. We suggest that the multiple copies of the stress-inducible hsp70 genes do not exist in the genome of D. melanogaster only to produce large amounts of the Hsp70 rapidly and at short notice, but that they are specifically regulated in a developmental-stage-specific manner. It is likely that the cost/benefit ratio of not producing or of producing a defined amount of Hsp70 under stress conditions varies for different cell types and under different physiological conditions and, accordingly, specific regulatory mechanisms operating at the transcriptional and post-transcriptional levels have evolved.

Regulation of heat shock proteins, Hsp70 and Hsp64, in heat-shocked Malpighian tubules of Drosophila melanogaster larvae.

It is known from earlier studies that the heat shock (HS) response in Malpighian tubules (MTs) of Drosophila larvae is different from that in other tissues because instead of the Hsp70 and other common heat shock proteins, Hsp64 and certain other new proteins are induced immediately after HS. In the present study, we examined the kinetics of the synthesis of Hsp70 and Hsp64 immediately after HS and during recovery from HS by 35S-methionine labeling and Western blotting. In addition, we also examined the transcriptional activity of hsp70 genes in larval MT cells at different times after HS by in situ hybridization and Northern blotting. The HS-induced synthesis of Hsp64 ceased by 1 hour of recovery from the HS when synthesis of the Hsp70 commenced. Our results revealed that the induced synthesis of Hsp64 immediately after HS was dependent on new transcription. Although the levels of Hsp70 in MT cells rapidly increased after its synthesis began during recovery, the levels of Hsp64 remained unaltered irrespective of its new synthesis occurring during or after HS. Inhibition of new Hsp64 synthesis by transcriptional or translational inhibitors also did not affect the total amount of this protein in MTs. The Hsp64 polypeptides synthesized in response to HS are degraded rapidly. Apparently, the cells in MTs maintain a balance between new synthesis of Hsp64 and its turnover so that under all conditions a more or less constant level of this protein is maintained. Although the Hsp70 synthesis started only after 1 hour of recovery, the hsp70 genes were transcriptionally activated immediately after HS and they continued to transcribe till at least 4 hours after the HS. The hsp70 transcripts in MT cells that recovered for 2 hours or longer did not contain the 3' untranslated regions (UTRs), which may allow their longer stability and translatability at normal temperature. Synthesis of Hsp70 during recovery period was dependent on continuing transcription. Assessment of the beta-galactosidase activity in 2 transgenic lines carrying the LacZ reporter gene under hsp70 promoter and different lengths of the 5'UTR suggested that the delayed translation of hsp70 transcripts in MTs is probably regulated by some elements in the 5'UTR.

Developmental regulation and complex organization of the promoter of the non-coding hsr(omega) gene of Drosophila melanogaster.

The nucleus-limited large non-coding hsr(omega)-n RNA product of the 93D or the hsr(omega) gene of Drosophila melanogaster binds to a variety of RNA-binding proteins involved in nuclear RNA processing. We examined the developmental and heat shock induced expression of this gene by in situ hybridization of nonradioactively labelled riboprobe to cellular transcripts in intact embryos, larval and adult somatic tissues of wild type and an enhancer-trap line carrying the hsr(omega) 05241 allele due to insertion of a P-LacZ-rosy+ transposon at -130 bp position of the hsr(omega) promoter. We also examined LacZ expression in the enhancer-trap line and in two transgenic lines carrying different lengths of the hsr(omega) promoter upstream of the LacZ reporter. The hsr(omega) gene is expressed widely at all developmental stages; in later embryonic stages, its expression in the developing central nervous system was prominent. In spite of insertion of a big transposon in the promoter, expression of the hsr(omega) 05241 allele in the enhancer-trap line, as revealed by in situ hybridization to hsr(omega) transcripts in cells, was similar to that of the wild type allele in all the embryonic, larval and adult somatic tissues examined. Expression of the LacZ gene in this enhancer-trap line was similar to that of the hsr(omega) RNA in all diploid cell types in embryos and larvae but in the polytene cells, the LacZ gene did not express at all, neither during normal development nor after heat shock. Comparison of the expression patterns of hsr(omega) gene and those of the LacZ reporter gene under its various promoter regions in the enhancer-trap and transgenic lines revealed a complex pattern of regulation, which seems to be essential for its dynamically varying expression in diverse cell types.

Male sterility associated with overexpression of the noncoding hsromega gene in cyst cells of testis of Drosophila melanogaster.

Of the several noncoding transcripts produced by the hsromega gene of Drosophila melanogaster, the nucleus-limited >10-kb hsromega-n transcript colocalizes with heterogeneous nuclear RNA binding proteins (hnRNPs) to form fine nucleoplasmic omega speckles. Our earlier studies suggested that the noncoding hsromega-n transcripts dynamically regulate the distribution of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments. Here we show that a P transposon insertion in this gene's promoter (at -130 bp) in the hsromega05421; enhancer-trap line had no effect on viability or phenotype of males or females, but the insertion-homozygous males were sterile. Testes of hsromega05421; homozygous flies contained nonmotile sperms while their seminal vesicles were empty. RNA:RNA in situ hybridization showed that the somatic cyst cells in testes of the mutant male flies contained significantly higher amounts of hsromega-n transcripts, and unlike the characteristic fine omega speckles in other cell types they displayed large clusters of omega speckles as typically seen after heat shock. Two of the hnRNPs, viz. HRB87F and Hrb57A, which are expressed in cyst cells, also formed large clusters in these cells in parallel with the hsromega-n transcripts. A complete excision of the P transposon insertion restored male fertility as well as the fine-speckled pattern of omega speckles in the cyst cells. The in situ distribution patterns of these two hnRNPs and several other RNA-binding proteins (Hrp40, Hrb57A, S5, Sxl, SRp55 and Rb97D) were not affected by hsromega mutation in any of the meiotic stages in adult testes. The present studies, however, revealed an unexpected presence (in wild-type as well as mutant) of the functional form of Sxl in primary spermatocytes and an unusual distribution of HRB87F along the retracting spindle during anaphase telophase of the first meiotic division. It appears that the P transposon insertion in the promoter region causes a misregulated overexpression of hsromega in cyst cells, which in turn results in excessive sequestration of hnRNPs and formation of large clusters of omega speckles in these cell nuclei. The consequent limiting availability of hnRNPs is likely to trans-dominantly affect processing of other pre-mRNAs in cyst cells. We suggest that a compromise in the activity of cyst cells due to the aberrant hnRNP distribution is responsible for the failure of individualization of sperms in hsromega05421; mutant testes. These results further support a significant role of the noncoding hsromega-n transcripts in basic cellular activities, namely regulation of the availability of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments.

Omega speckles - a novel class of nuclear speckles containing hnRNPs associated with noncoding hsr-omega RNA in Drosophila.

Fluorescence RNA:RNA in situ hybridization studies in various larval and adult cell types of Drosophila melanogaster showed that the noncoding hsr-omega nuclear (hsromega-n) transcripts were present in the form of many small speckles. These speckles, which we name 'omega speckles', were distributed in the interchromatin space in close proximity to the chromatin. The only chromosomal site where hsromega-n transcripts localized was the 93D locus or the hsromega gene itself. The number of nucleoplasmic speckles varied in different cell types. Heat shock, which inhibits general chromosomal transcription, caused the individual speckles to coalesce into larger but fewer clusters. In extreme cases, only a single large cluster of hsromega-n transcripts localizing to the hsromega locus was seen in each nucleus. In situ immunocytochemical staining using antibodies against heterogenous nuclear RNA binding proteins (hnRNPs) like HRB87F, Hrp40, Hrb57A and S5 revealed that, in all cell types, all the hnRNPs gave a diffuse staining of chromatin areas and in addition, were present as large numbers of speckles. Colocalization studies revealed an absolute colocalization of the hnRNPs and the omegaspeckles. Heat shock caused all the hnRNPs to cluster together exactly, following the hsromega-n transcripts. Immunoprecipitation studies using the hnRNP antibodies further demonstrated a physical association of hnRNPs and hsromega transcripts. The omegaspeckles are distinct from interchromatin granules since nuclear speckles containing serine/arginine-rich SR-proteins like SC35 and SRp55 did not colocalize with the &ohgr; speckles. The speckled distribution of hnRNPs was completely disrupted in hsromega nullosomics. We conclude that the hsromega-n transcripts play essential structural and functional roles in organizing and establishing the hnRNP-containing omega speckles and thus regulate the trafficking and availability of hnRNPs and other related RNA binding proteins in the cell nucleus.