Enhanced oral delivery of celecoxib via the development of a supersaturable amorphous formulation utilising mesoporous silica and co-loaded HPMCAS.

Stabilization of amorphous formulations via mesoporous silica has gained considerable attention for oral delivery of poorly soluble drugs. The release of the drug from the silica is expected to generate supersaturation which is often associated with subsequent precipitation. The aim of the study was hence to develop a novel supersaturable amorphous formulation through the co-loading of a BCS class II drug Celecoxib (CXB) with a precipitation inhibitor hydroxypropyl methylcellulose acetate succinate (HPMCAS) onto the silica. The addition of HPMCAS did not hamper the adsorption but on the contrary promoted the complete solid state conversion of the drug as proved by DSC analysis. In an in vitro pH shift assay, the CXB-HPMCAS co-loaded silica achieved a 5-fold solubility increase over the crystalline CXB and over the CXB-loaded silica blended with HPMCAS which did not show any enhancement. The drug co-loaded silica was then suspended in an aqueous vehicle facilitating the dosing to animals. The CXB-HPMCAS co-loaded silica suspension achieved 15-fold solubility increase in vitro over the crystalline counterpart which translated in 1.35-fold Cmax increase in vivo after oral dosing in rats. This approach represents a novel formulation strategy to maximize in vivo exposure of poorly soluble drugs critical for discovery studies.

Human aortic smooth muscle cells are insulin resistant at the receptor level but sensitive to IGF1 and IGF2.

Whether insulin, at physiological concentrations, has direct effects on vascular smooth muscle cells (VSMCs) remains controversial. Our aim was to characterize the mechanism for insulin resistance in VSMCs. For comparison, the effects of IGF1 and IGF2 were also studied. Cultured human aortic smooth muscle cells (HASMC) were used. Receptor mRNA was analyzed by quantitative reverse transcription PCR and receptor protein by ELISA and western blot. Biological effects were studied by thymidine incorporation and glucose accumulation. In HASMC, both mRNA and protein expression of IGF1 receptors (IGF1R) were fivefold higher compared to insulin receptor (IR). IR isoform A mRNA was 13-fold more expressed than IR isoform B. IR and IGF1R co-precipitated, indicating the presence of hybrid IR/IGF1R. Phosphorylation of the IGF1R beta-subunit was obtained by IGF1 10(-9)-10(-8) mol/l and IGF2 10(-8) mol/l. IR beta-subunit was phosphorylated by IGF1 10(-8) mol/l but not by insulin. IGF1 stimulated IR substrate-1 and AKT at 10(-8) mol/l and extracellular signal-regulated kinases 1 and 2 at 10(-9)-10(-8) mol/l respectively. IGF1 and 2 at a concentration of 10(-8)-10(-7) mol/l significantly stimulated (3)H-thymidine incorporation, whereas insulin did not. (14)C-Glucose accumulation was stimulated by IGF1 or IGF2 10(-8)-10(-7) mol/l, and also by insulin 10(-7) mol/l. Our results suggest that IGF1R and hybrid IR/IGF1R are activated by physiological concentrations of IGF1 and 2 in HASMC and this propagates downstream signaling and biological effects, while insulin has no effect on its receptor or downstream signaling probably due to a preponderance of IGF1R and incorporation of IR into hybrid IR/IGF1R.

Characterization of tryptamine 5-hydroxylase and serotonin synthesis in rice plants.

Serotonin is a well-known pineal hormone that in mammals plays a key role in mood. In plants, serotonin is implicated in several physiological roles such as flowering, morphogenesis, and adaptation to environmental changes. However, its biosynthetic enzyme in plants has not been characterized. Therefore, we measured the serotonin content and enzyme activity responsible for serotonin biosynthesis in rice seedlings. Tryptamine 5-hydroxylase (T5H), which converts tryptamine into serotonin, was found as a soluble enzyme that had maximal activity in the roots. The maximal activity of T5H was closely associated with the enriched synthesis of serotonin in roots. Tetrahydropterine-dependent T5H activity was inhibited by tyramine, tryptophan, 5-OH-tryptophan, and octopamine, but remained unaltered by dopamine in vitro. The tissues of rice seedlings grown in the presence of tryptamine exhibited a dose-dependent increase in serotonin in parallel with enhanced T5H enzyme activity. However, no significant increase in serotonin was observed in rice tissues grown in the presence of tryptophan, suggesting that tryptamine is a bottleneck intermediate substrate for serotonin synthesis.

Partial characterization of farnesyl and geranylgeranyl diphosphatases induced in rice seedlings by UV-C irradiation.

This study reports the identification and characterization of allyl diphosphatases (EC 3.1.7.1) in plants by using rice seedlings. Two distinct Mg(2+)-independent allyl diphosphatases, which convert farnesyl diphosphate (FDP) and geranylgeranyl diphosphate (GGDP) into farnesol and geranylgeraniol, respectively, were induced in rice seedlings irradiated with UV-C. Farnesyl diphosphatase (FDPase) and geranylgeranyl diphosphatase (GGDPase) are located in the microsomal fraction. The relative specific activity of FDPase was much higher than the specific activity of GGDPase. FDPase activity was inhibited by GGDP (50% inhibition at 5 microM) and geranyl diphosphate (50% inhibition at 100 microM). In contrast, GGDPase activity was inhibited 50% by 50 microM isopentenyl diphosphate or 100 microM FDP. The optimal pH for FDPase was 6.3 and for GGDPase was 7.9.

Cloning and characterization of a hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase induced in response to UV-C and wounding from Capsicum annuum.

Hydroxycinnamoyl-CoA : tyramine N-(hydroxycinnamoyl) transferase (THT) is a pivotal enzyme in the synthesis of N-(hydroxycinnamoyl)-amines, which are associated with cell wall fortification in plants. The cDNA encoding THT was cloned from the leaves of UV-C treated Capsicum annuum (hot pepper) using a differential screening strategy. The predicted protein encoded by the THT cDNA is 250 amino acids in length and has a relative molecular mass of 28,221. The protein sequence derived from the cDNA shares 76% and 67% identity with the potato and tobacco THT protein sequences, respectively. The recombinant pepper THT enzyme was purified using a bacterial overexpression system. The purified enzyme has a broad substrate specificity including acyl donors such as cinnamoyl-, sinapoyl-, feruloyl-, caffeoyl-, and 4-coumaroyl-CoA and acceptors such as tyramine and octopamine. In UV-C treated plants, the THT mRNA was strongly induced in leaves, and the elevated level of expression was stable for up to 36 h. THT mRNA also increased in leaves that were detached from the plant but not treated with UV-C. THT expression was measured in different plant tissues, and was constitutive at a similar level in leaf, root, stem, flower and fruit. Induction of THT mRNA was correlated with an increase in THT protein.

Molecular cloning and cultivar specific expression of MAP kinases from Capsicum annuum.

Two MAP kinases, MK1 and MK2, were cloned from Capsicum annuum (pepper) cv. Subicho using a parsley MAP kinase gene as a heterologous probe. MK1 and MK2 encode stress-inducible protein kinases that can contribute to the response to wounding, UV-C, and cold. MK1 has a 92% amino acid identity with WIPK of tobacco. It was transcriptionally induced in response to wounding. In contrast, no detectable MK1 transcript was found in unwounded leaves of pepper. MK2 has a high level of amino acid homology to MAP kinases, such as NTF4 and SIPK and was constitutively expressed in all tissues. Both MK transcripts were downregulated by UV-C treatment. Each MK protein activation was independently wound-inducible in a cultivar dependent manner. MK1 is phosphorylated in cv. Pungchon but not cv. Subicho; whereas, the MK2 protein activation by wounding is restricted to cv. Subicho. In addition, de novo synthesis of the MK1 protein and tyrosine phosphorylation was rapidly and transiently induced in cv. Pungchon by wounding. In contrast, it is highly unlikely that the MK1 protein is produced in cv. Subicho, even though there is an abundant expression of MK1 mRNA after wounding in this cultivar. In Escherichia coli, which overexpresses MK1, autophosphorylation is observed at conserved threonine and tyrosine phosphorylation sites.

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen.

Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.

Cloning of a sesquiterpene cyclase and its functional expression by domain swapping strategy.

Sesquiterpene cyclase, the first committed step enzyme from the general isoprenoid building block farnesyl pyrophosphate (FPP) for the synthesis of phytoalexin capsidiol, was isolated from the UV-C treated leaves of Capsicum annuum. This sesquiterpene cyclase, termed as CASC2 showing 77% amino acid identity with the previously cloned sesquiterpene cyclase CASC1, was composed of 560 amino acids with a calculated molecular mass of 64,907. The mRNA expression pattern of CASC2 was very similar to that of CASC1 during the time course of UV-C irradiated leaves of pepper on RNA blot analysis by using each specific probe. The heterologous expression in Escherichia coli using the CASC2 full length failed; however the chimeric construct of CASC2 in which the amino terminal 164 amino acid substituted by the equivalent portion of either CASC1 or tobacco sesquiterpene cyclase was capable of expressing the functional sesquiterpene cyclase activities. The radio-labeled enzymatic products catalyzed by the partially purified chimeric CASC2 were comigrated with authentic radio-labeled sesquiterpene on thin layer chromatography.

Managed care and the implementation of the State Children's Health Insurance Program.

More than two years after its enactment, the Health Care Financing Administration, Baltimore, issued proposed regulations implementing the State Children's Health Insurance Program (SCHIP), enacted under the Balanced Budget Act of 1997. This program was enacted to provide health insurance coverage to the estimated 10 million uninsured children living in the United States. This article provides an overview of SCHIP and how it has been implemented in the various states.

Cloning and bacterial expression of sesquiterpene cyclase, a key branch point enzyme for the synthesis of sesquiterpenoid phytoalexin capsidiol in UV-challenged leaves of Capsicum annuum.

Sesquiterpene cyclase, a branch point enzyme in the general isoprenoid pathway for the synthesis of phytoalexin capsidiol, was induced in detached leaves of Capsicum annuum (pepper) by UV treatment. The inducibility of cyclase enzyme activities paralleled the absolute amount of cyclase protein(s) of pepper immunodetected by monoclonal antibodies raised against tobacco sesquiterpene cyclase. A cDNA library was constructed with poly(A)+ RNA isolated from 24 h UV-challenged leaves of pepper. A cDNA clone for sesquiterpene cyclase in pepper was isolated by using a tobacco 5-epi aristolochene synthase gene as a heterologous probe. The predicted protein encoded by this cDNA was comprised of 559 amino acids and had a relative molecular mass of 65,095. The primary structural information from the cDNA clone revealed that it shared 77%, 72% and 49% identity with 5-epi aristolochene, vetispiradiene, and cadinene synthase, respectively. The enzymatic product catalyzed by the cDNA clone in bacteria was identified as 5-epi aristolochene, as judged by argentation TLC. RNA blot hybridization demonstrated the induction of an mRNA consistent with the induction of cyclase enzyme activity in UV-treated pepper.

Molecular characterization of tobacco squalene synthase and regulation in response to fungal elicitor.

The enzyme squalene synthase (SS) represents the first commitment of carbon from the general isoprenoid pathway toward sterol biosynthesis and is a potential point for regulation of sterol biosynthesis. The isolation and characterization of tobacco (Nicotiana tabacum) squalene synthase (TSS) cDNA and genomic DNA clones, as well as determination of the steady state level of TSS mRNA in response to elicitor treatment, were investigated. cDNA clones for TSS were isolated from poly (A)+ RNA using a reverse transcription/polymerase chain reaction (RT/PCR) method. A 1233-bp cDNA clone was generated that contained an open reading frame of 411 amino acids giving a predicted molecular mass of 46.9 kDa. Comparison of the TSS deduced amino acid sequence with currently described SS from different species showed the highest identify with Nicotiana benthamiana (97%), followed by Glycyrrhiza glabra (81%), Arabidopsis thaliana (74%), rat (40%), and yeast (37%). Expression of a soluble form of the TSS enzyme with enzymatic activity in Escherichia coli was achieved by truncating 24 hydrophobic amino acids at the carboxy terminus. Characterization of genomic TSS (gTSS) revealed a gene of 7.086 kb with a complex organization of small exons and large introns not typical of plant genes. Southern blot hybridization indicated only two copies of the SS gene in the tobacco genome. Treatment of tobacco cell suspension cultures with a fungal elicitor dramatically reduced TSS enzymatic activity, lowering it to zero within 24 h. Analysis of TSS mRNA levels, by RNA blot hybridization and primer extension assays, in elicitor-treated cells indicated that the transcript level remained largely unchanged over this 24-h period. These results suggest that the suppression of TSS enzyme activity in elicitor-treated cells may result from a posttranscriptional modification of TSS.

Regulation of sesquiterpene cyclase gene expression. Characterization of an elicitor- and pathogen-inducible promoter.

The promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene, a key regulatory step in sesquiterpene phytoalexin biosynthesis, has been analyzed. The EAS4 promoter was fused to the beta-glucuronidase (GUS) reporter gene, and the temporal and spatial expression patterns of GUS activity were examined in stably transformed plants and in transient expression assays using electroporated protoplasts of tobacco. No GUS activity was observed in any tissues under normal growth conditions. A low level of GUS activity was detected in wounded leaf, root, and stem tissues, whereas a much higher level was observed when these tissues were challenged with elicitors or microbial pathogens. The GUS expression pattern directed by the EAS4 promoter was identical to the induction patterns observed for the endogenous sesquiterpene cyclase genes. Neither exogenous salicylic acid nor methyl jasmonate induced GUS expression; and H2O2 induced GUS expression to only a limited extent. Although the EAS4 promoter contains cis-sequences resembling previously identified transcriptional control motifs, other cis-sequences important for quantitative and qualitative gene expression were identified by deletion and gain-of-function analyses. The EAS4 promoter differs from previously described pathogen-/elicitor-inducible promoters because it only supports inducible gene expression and directs unique spatial expression patterns.

Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase.

Terpene cyclases catalyze the synthesis of cyclic terpenes with 10-, 15-, and 20-carbon acyclic isoprenoid diphosphates as substrates. Plants have been a source of these natural products by providing a homologous set of terpene synthases. The crystal structures of 5-epi-aristolochene synthase, a sesquiterpene cyclase from tobacco, alone and complexed separately with two farnesyl diphosphate analogs were analyzed. These structures reveal an unexpected enzymatic mechanism for the synthesis of the bicyclic product, 5-epi-aristolochene, and provide a basis for understanding the stereochemical selectivity displayed by other cyclases in the biosynthesis of pharmacologically important cyclic terpenes. As such, these structures provide templates for the engineering of novel terpene cyclases.

Pre-steady-state study of recombinant sesquiterpene cyclases.

An Escherichia coli expression system was used to generate hexahistidyl-tagged plant sesquiterpene cyclases, which were readily purified by a single affinity chromatographic step. Genes for Hyoscyamus muticus vetispiradiene synthase (HVS), a chimeric 5-epi-aristolochene synthase (CH3), and a chimeric sesquiterpene cyclase possessing multifunctional epi-aristolochene and vetispiradiene activity (CH4) were expressed in bacterial cells, which resulted in the sesquiterpene cyclases accumulating to 50% of the total protein and 35% of the soluble protein. From initial velocity experiments, the Michaelis constant for HVS was 3.5 microM, while CH3 and CH4 exhibited smaller values of 0.7 and 0.4 microM, respectively. Steady-state catalytic constants were from 0.02 to 0.04 s-1. A combination of pre-steady-state rapid quench experiments, isotope trapping experiments, and experiments to measure the burst rate constant as a function of substrate concentration revealed that turnover in all three cyclases is limited by a step after the initial chemical step involving rupture of the carbon-oxygen bond in farnesyl diphosphate (FPP). Rate constants for the limiting step were 10-70-fold smaller than for the initial chemical step. Dissociation constants for the enzyme-substrate complex (20-70 microM) were determined from the pre-steady-state experiments and were significantly larger than the observed Michaelis constants. A mechanism that involves an initial, rapid equilibration of enzyme with substrate to form an enzyme-substrate complex, followed by a slower conversion of FPP to an enzyme-bound hydrocarbon and a subsequent rate-limiting step, is proposed for the three enzymes. Interestingly, the multifunctional chimeric enzyme CH4 exhibited both a tighter binding of FPP and a faster conversion of FPP to products than either of its wild-type parents.

Identifying functional domains within terpene cyclases using a domain-swapping strategy.

Cyclic terpenes and terpenoids are found throughout nature. They comprise an especially important class of compounds from plants that mediate plant- environment interactions, and they serve as pharmaceutical agents with antimicrobial and anti-tumor activities. Molecular comparisons of several terpene cyclases, the key enzymes responsible for the multistep cyclization of C10, C15, and C20 allylic diphosphate substrates, have revealed a striking level of sequence similarity and conservation of exon position and size within the genes. Functional domains responsible for a terminal enzymatic step were identified by swapping regions approximating exons between a Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) gene and a Hyoscyamus muticus vetispiradiene synthase (HVS) gene and by characterization of the resulting chimeric enzymes expressed in bacteria. While exon 4 of the TEAS gene conferred specificity for the predominant reaction products of the tobacco enzyme, exon 6 of the HVS gene conferred specificity for the predominant reaction products of the Hyoscyamus enzyme. Combining these two functional domains of the TEAS and HVS genes resulted in a novel enzyme capable of synthesizing reaction products reflective of both parent enzymes. The relative ratio of the TEAS and HVS reaction products was also influenced by the source of exon 5 present in the new chimeric enzymes. The association of catalytic activities with conserved but separate exonic domains suggests a general means for generating additional novel terpene cyclases.

Cloning and bacterial expression of a sesquiterpene cyclase from Hyoscyamus muticus and its molecular comparison to related terpene cyclases.

Genomic and cDNA clones for vetispiradiene synthase, a sesquiterpene cyclase found in Hyoscyamus muticus, were isolated using a combination of reverse transcription-polymerase chain reactions and conventional cloning procedures. RNA blot hybridization demonstrated an induction of mRNA consistent with the induction of cyclase enzyme activity in elicitor-treated cells, DNA blot hybridization indicated a gene family of 6 to 8 members, and bacterial expression of 3 cDNA clones indicated that each coded for a vetispiradiene synthase enzyme activity catalyzing the synthesis of a single reaction product. Intron-exon organization of the vetispiradiene synthase gene was identical with that previously described for 5-epi-aristolochene synthase (tobacco sesquiterpene cyclase) and casbene synthase (castor bean diterpene cyclase), and the vetispiradiene synthase amino acid sequence was 77% identical with and 81% similar to the tobacco sesquiterpene cyclase. Regions of the vetispiradiene synthase sequence centered around amino acids 60, 100, and 370 were conspicuously different relative to the tobacco sesquiterpene cyclase. The sequence similarity between the tobacco and H. muticus enzymes is suggested to be reflective of the conservation of several partial reactions common to both enzymes, and the differences may be reflective of a partial reaction unique to each enzyme.

Expression of a plant sesquiterpene cyclase gene in Escherichia coli.

5-Epi-aristolochene synthase is a sesquiterpene cyclase activity found in pathogen-challenged tobacco cells, but not in nonchallenged tissues, and appears to be encoded by a complex gene family. As a prerequisite to assessing the functional significance of these multiple genes, bacterial expression systems were examined for their capacity to express a tobacco sesquiterpene cyclase cDNA. Insertion of full-length 5-epi-aristolochene synthase cDNA into two commonly used expression vectors, pET-11d and pGBT-T19, resulted in high level expression of the cyclase activity. The highest level of expression occurred 3 h after induction with low concentrations (0.1-0.5 mM) of IPTG, incubation at 27 degrees C instead of 37 degrees C, and in the bacterial host strain BL21(DE3). Under these conditions, the cyclase protein constituted 5 to 8% of the soluble and 35% of the total Escherichia coli proteins. Enzyme reaction products of the native tobacco and recombinant enzyme were identical, based on argentation-thin layer chromatography. Deletion mutants of the cyclase gene corresponding to the amino and carboxy termini of the enzyme were prepared. The cyclase proteins resulting from bacterial expression of these mutant constructs were found largely in the insoluble protein fraction and no soluble enzyme activity was detected.

Mental health among immigrant and refugee children of divorced parents.

The mental status of 27 children in divorced immigrant families and 17 children in divorced refugee families was examined, and compared to that of 113 children in divorced Swedish families. Differences in divorce-pattern between these families and Swedish divorced families were analysed. Viewed together, the immigrant and refugee children displayed a significantly higher symptom load compared to Swedish children from both divorced and intact homes. When examined separately, the refugee children but not the immigrant children were more troubled than Swedish children from divorced families. The shorter time the children had spent in Sweden, the higher was their symptom load. In comparison to Swedish divorced couples, the immigrant and refugee couples had been married for a shorter time, had been unhappy for a longer time prior to divorce, and joint custody was less common. Most of the marriages had been unhappy before the arrival in Sweden, but a dissolution had not been seen as feasible earlier. As among the Swedish couples, it was the woman who had initiated the divorce in most cases, and it was also the mother who became the residential parent in most cases.

Long-term factors in the psychology of fertility.

PIP: Population psychology has generally concentrated upon a micro and rational approach in the study of individual decisions related to fertility. The psychological theory of fertility is concerned primarily with a short-time perspective in which parents consciously decide to have offspring or simply respond to their biological urges of the moment for intercourse. The author expands this view by concentrating upon the concept of lineage consciousness, manifestations of its influence and presence in current society, and its relation to fertility. He begins by discussing the relative neglect of linkages between individual and social concerns and suggests the importance of often overlooked long-range motivations. He then describes the current social and technical changes which permit identification and analysis of background conditions to decision making. Social identity and consciousness of one's mortality are discussed and connected with psychological and sociological conditions. Theoretical insights are finally shown in relation to research and practical applications.^ieng