Polymer incorporation method affects the physical stability of amorphous indomethacin in aqueous suspension.

This study reports the potential of different polymers and polymer incorporation methods to inhibit crystallisation and maintain supersaturation of amorphous indomethacin (IND) in aqueous suspensions during storage. Three different polymers (poly(vinyl pyrrolidone) (PVP), hydroxypropyl methylcellulose (HPMC) and Soluplus® (SP)) were used and included in the suspensions either as a solid dispersion (SD) with IND or dissolved in the suspension medium prior to the addition of amorphous IND. The total concentrations of both IND and the polymer in the suspensions were kept the same for both methods of polymer incorporation. All the polymers (with both incorporation methods) inhibited crystallisation of the amorphous IND. The SDs were better than the predissolved polymer solutions at inhibiting crystallisation. The SDs were also better at maintaining drug supersaturation. SP showed a higher IND crystallisation inhibition and supersaturation potential than the other polymers. However, this depended on the method of addition. IND in SD with SP did not crystallise, nor did the SD generate any drug supersaturation, whereas IND in the corresponding predissolved SP solution crystallised (into the recently characterised η polymorphic form of the drug) but also led to a more than 20-fold higher IND solution concentration than that observed for crystalline IND. The ranking of the polymers with respect to crystallisation inhibition potential in SDs was SP≫PVP>HPMC. Overall, this study showed that both polymer type and polymer incorporation method strongly impact amorphous form stability and drug supersaturation in aqueous suspensions.

Stroboscopic supercontinuum white-light interferometer for MEMS characterization.

We used a supercontinuum-based scanning white-light interferometer to characterize the oscillation of a MEMS device. The output of a commercially available supercontinuum light source (FiberWare Ilum II USB) was modulated to achieve stroboscopic operation. By synchronizing the modulation frequency of the source to the sample oscillation, dynamic 3-D profile measurements were recorded. These results were validated against those obtained with a white light LED setup. The measured maximum deflection of a 400×25×4 µm(3) microbridge driven with 0-6.8 V sinusoidal voltage at 10 Hz was 1.42±0.03 µm (supercontinuum), which agreed with the LED measurement. The method shows promise for characterization of high-frequency MEMS devices.

Impact of maternal diet during pregnancy and breastfeeding on infant metabolic programming: a prospective randomized controlled study.

OBJECTIVES: To evaluate the impact of maternal diet and intensive dietary counselling during pregnancy and breastfeeding on the infant's metabolic status. SUBJECTS/METHODS: At the first trimester of pregnancy, 256 women were randomized into a control/placebo group and two dietary counselling groups (diet/probiotics and diet/placebo). The counselling, with double-blind randomization to probiotics (Lactobacillus rhamnosus GG and Bifidobacterium lactis) or placebo, targeted excessive saturated fat and low fibre consumption. Maternal diet was evaluated repeatedly during pregnancy and postpartum by means of 3 days' food diaries. Metabolic markers, serum 32-33 split and intact proinsulin, leptin/adiponectin ratio, skinfold thickness and waist circumference were measured of 194 healthy infants at the age of 6 months, and the high levels were taken to mirror adverse metabolic status. RESULTS: The proportion of infants with a high 32-33 split proinsulin was significantly lower in dietary counselling with probiotics (n = 6/62, 9.7%) or placebo (n = 7/69, 10.1%) compared with the control/placebo group (n = 17/63, 27.0%). The high split proinsulin was associated with larger skinfold thickness, waist circumference and higher leptin/adiponectin ratio in the infants (P < 0.05). With respect to maternal diet during pregnancy, the highest and lowest tertiles of fat intake increased the infant's risk of high split proinsulin, whereas those of butter associated correspondingly with the infant's waist circumference. Further, breastfed infants showed a reduced risk of high split proinsulin and leptin/adiponectin ratio compared with formula-fed infants. CONCLUSIONS: Modification of maternal diet during pregnancy and breastfeeding may benefit infant metabolic health. High split proinsulin reflects adverse metabolic status in infancy, which can be improved by early dietary counselling.

Using modified Kramers-Kronig relations to test transmission spectra of porous media in THz-TDS.

We show that modified Kramers-Kronig relations provide a useful tool to test the validity of the complex refractive index extracted from transmission terahertz spectra of porous matrices containing pharmaceutical materials. The role of scattering of terahertz radiation is qualitatively considered as a reason for the observed discrepancy between experimental data and the values extracted from the inverted complex refractive index. As an example we present an analysis of the terahertz spectra of carbamazepine and lactose alpha-monohydrate.

Better understanding of dissolution behaviour of amorphous drugs by in situ solid-state analysis using Raman spectroscopy.

Amorphous drugs have a higher kinetic solubility and dissolution rate than their crystalline counterparts. However, this advantage is lost if the amorphous form converts to the stable crystalline form during the dissolution as the dissolution rate will gradually change to that of the crystalline form. The purpose of this study was to use in situ Raman spectroscopy in combination with either partial least squares discriminant analysis (PLS-DA) or partial least squares (PLS) regression analysis to monitor as well as quantify the solid-phase transitions that take place during the dissolution of two amorphous drugs, indomethacin (IMC) and carbamazepine (CBZ). The dissolution rate was higher from amorphous IMC compared to the crystalline alpha- and gamma-forms. However, the dissolution rate started to slow down during the experiment. In situ Raman analysis verified that at that time point the sample started to crystallize to the alpha-form. Amorphous CBZ instantly started to crystallize upon contact with the dissolution medium. The transition from the amorphous form to CBZ dihydrate appears to go through the anhydrate form I. Based on the PLS analysis the amount of form I formed in the sample during the dissolution affected the dissolution rate. Raman spectroscopy combined with PLS-DA was also more sensitive to the solid-state changes than X-ray powder diffraction (XRPD) and was able to detect changes in the solid-state that could not be detected with XRPD.

Biosynthesis of 32P-labelled hydroxocobalamin and a study of its behaviour in rats.

Using Propionibacterium freudenreichii and 32P-ATP, batches of 32P-labelled cobalamin (Cbl) were biosynthesized with a maximum specific activity of 61 microCi/mg, i.e. about 100 times higher than previously reported. Pharmacological doses mixed with 57Co-Cbl were injected subcutaneously in the form of hydroxo-Cbl into rats subsequently killed 5-20 days later. The two labelled Cbls were distributed in approximately the same way, the highest concentration being found in kidney (typical for rats) and about one-fifth of that in liver. These findings tallied with previous observations with radioactive cyano-Cbl and microbiological assay. In all injected rats, the 57Co/32P ratio was lower in liver than in kidney. Drugs eradicating the intestinal flora had no influence. In rats receiving the vitamin orally, the ratio was higher in liver than in kidney. All of our findings could be due to formation of a cobinamide-like compound lacking phosphorus. It is concluded that we have produced radiophosphorus-labelled Cbl that enables studies in vivo.

Software algorithm for automatic interpretation of mass spectra of glycerolipids.

A new software algorithm for automatic interpretation of mass spectra of glycerolipids has been developed. The algorithm utilizes a user-specified list of parameters needed to process the spectra. The compounds in mass spectra are identified according to range of measured m/z values, after which the spectra are automatically corrected by the content of naturally occurring isotopes and ion intensities of identified compounds by response correction factors. Automatic processing of the spectra was shown to be accurate and reliable by testing with numerous spectra of glycerophospholipids obtained by liquid chromatography/electrospray ionization mass spectrometry and by comparing the results with manual interpretation of the spectra. If quantitative analysis using internal standards is performed, all the identified compounds in the sample are quantified automatically. A dilution factor may be defined for each sample and is applied to correct the alterations in sample concentration during sample preparation. Processing of several replicate spectra simultaneously produces mean results with standard deviations. The software may also be used to subtract the results of two analyses and to calculate the mean result of replicate subtractions. The algorithm was shown to save time and labor in repetitive processing of mass spectra of similar type. It may be applied to processing of spectra obtained by various mass spectrometric methods.

Duration of untreated psychosis and its correlates in first-episode psychosis in Finland and Spain.

OBJECTIVE: To examine the association of duration of untreated psychosis (DUP) with early course characteristics in first-episode psychosis in Finland and Spain. METHOD: Eighty-six patients from Finland (49) and Spain (37) were evaluated on various early course characteristics. RESULTS: The mean value of DUP was 4.0 months (median 2 months) for the Finnish patients and 9.9 months (median 2 months) for the Spanish ones. In both groups, long DUP was associated with insidious onset, poor global functioning, and laboral incapability. Among the Finnish patients exclusively, long DUP correlated with a weak earlier social network, instability of professional identity, long duration of prodromal symptoms, psychological dependency on the family, and criticism by the parents of the patient. Among the Spanish patients only, longer DUP was associated with more severe positive symptoms at admission. CONCLUSION: There are universal psychosocial factors influencing DUP, but also cultural differences may have an impact on the treatment delay.

cDNA cloning, expression studies and chromosome mapping of human type I serine/threonine kinase receptor ALK7 (ACVR1C).

Transforming growth factor-beta (TGF-beta) superfamily related growth factors signal by binding to transmembrane type I and type II receptor serine/threonine kinases (RSTK), which phosphorylate intracellular Smad transcription factors in response to ligand binding. Here we describe the cloning of the human type I RSTK activin receptor-like kinase 7 (ALK7), an orthologue of the previously identified rat ALK7. Nodal, a TGF-beta member expressed during embryonic development and implicated in developmental events like mesoderm formation and left-right axis specification, was recently shown to signal through ALK7. We found ALK7 mRNA to be most abundantly expressed in human brain, pancreas and colon. A cDNA encoding the open reading frame of ALK7 was obtained from a human brain cDNA library. Furthermore, a P1 artificial chromosome (PAC) clone containing the human ALK7 gene was isolated and fluorescent in situ hybridization (FISH) on metaphase chromosomes identified the gene locus as chromosome 2q24.1-->q3. To test the functionality of the ALK7 signaling, we generated recombinant adenoviruses containing a constitutively active form of ALK7 (Ad-caALK7), which is capable of activating downstream targets in a ligand independent manner. Infection with Ad-caALK7 of MIN6 insulinoma cells, in which ALK7 has previously been shown to be endogenously expressed, led to a marked increase in the phosphorylation of Smad2, a signaling molecule also used by TGF-betas and activins.

Two-year outcome in first-episode psychosis treated according to an integrated model. Is immediate neuroleptisation always needed?

In this multicentre study the two-year outcome of two groups of consecutive patients (total N = 106) with first-episode functional non-affective psychosis, both treated according to the 'need-specific Finnish model', which stresses teamwork, patient and family participation and basic psychotherapeutic attitudes, was compared. No alternative treatment facilities were available in the study sites. The two study groups differed in the use of neuroleptics: three of the sites (the experimental group) used a minimal neuroleptic regime whilst the other three (the control group) used neuroleptics according to the usual practice. Total time spent in hospital, occurrence of psychotic symptoms during the last follow-up year, employment, GAS score and the Grip on Life assessment were used as outcome measures. In the experimental group 42.9% of the patients did not receive neuroleptics at all during the whole two-year period, while the corresponding proportion in the control group was 5.9%. The overall outcome of the whole group could be seen as rather favourable. The main result was that the outcome of the experimental group was equal or even somewhat better than that of the control group, also after controlling for age, gender and diagnosis. This indicates that an integrated approach, stressing intensive psychosocial measures, is recommended in the treatment of acute first-episode psychosis.

Mutations in the AIRE gene: effects on subcellular location and transactivation function of the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy protein.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal disease with recessive inheritance. It is characterized by multiple autoimmune endocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The defective gene responsible for this disease was recently isolated, and several different mutations in the novel gene, AIRE, have been identified, by us and by others, in patients with APECED. We have shown that the APECED protein is mainly localized, both in vitro and in vivo, to the cell nucleus, where it forms distinct speckles. This accords with the predicted structural features of the protein, which suggest involvement of AIRE in the regulation of gene transcription. Here, we report the results of mutational analyses of a series of 112 patients with APECED who were from various ethnic backgrounds. A total of 16 different mutations, covering 91% of disease alleles, were observed; of these, 8 were novel. The mutations are spread throughout the coding region of AIRE, yet four evident mutational hotspots were observed. In vitro expression of four different naturally occurring nonsense and missense mutations revealed a dramatically altered subcellular location of the protein in cultured cells. Interestingly, the wild-type APECED protein tethered to the Gal4 DNA-binding domain acted as a strong transcriptional activator of reporter genes in mammalian cells, whereas most of the analyzed mutant polypeptides had lost this capacity.

Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B.

Although targeted gene disruption of GDF-9, an oocyte derived growth factor, leads to an arrest of folliculogenesis and causes infertility in female mice, little is known on the expression of GDF-9 protein in the ovary. We show that GDF-9 protein is expressed in rat oocytes during folliculogenesis from the early primary follicle stage onwards but the most intensive immunostaining was seen in primary and preantral follicles. Northern blot analyses of the ontogeny of GDF-9 gene expression in postnatal rat ovaries showed that the GDF-9 transcript levels are clearly increased on the second postnatal day concomitant with the appearance of primary follicles. Interestingly, Northern blot and in situ hybridization analyses indicate a similar expression pattern for GDF-9B, the rat ortholog of a mouse GDF-9 like factor for which we recently reported the partial amino acid sequence. The polypeptide sequences deduced from isolated ovarian cDNAs indicate that the rat GDF-9 prepropeptide is 440 amino acids (aa) in length and the putative mature peptide is 135 aa whereas rat GDF-9B is 391 aa long and the mature region is 125 aa. We conclude that (1) the GDF-9 protein is highly expressed in the oocytes of primary follicles of rat ovaries suggesting that it plays a role mainly in early folliculogenesis and that (2) the full-length polypeptide sequence of GDF-9B suggests that this novel TGF-beta family member is likely to be a secreted growth factor that may regulate folliculogenesis at similar developmental stages as GDF-9.

Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis.

Growth differentiation factor 9 (GDF-9) is a transforming growth factor-beta family member that is required for normal folliculogenesis in female mice, but its role as a regulator of human fertility is still unclear. We determined here by in situ hybridization and immunohistochemical analyses the localization of the GDF-9 messenger ribonucleic acid (mRNA) and protein during human folliculogenesis. The GDF-9 transcripts were not detected in primordial follicles, but they are abundantly expressed in primary follicles in frozen sections of ovarian cortical tissue material obtained at laparoscopic surgery. We raised antipeptide antibodies against GDF-9 and showed by immunohistochemical studies on paraffin sections of whole human ovaries that the GDF-9 protein is most abundantly expressed in primary follicles. We recently demonstrated that a novel GDF-9-related factor, GDF-9B, is coexpressed with GDF-9 during murine folliculogenesis. We now isolated human GDF-9B complementary DNA and genomic clones and report the unusually restricted expression pattern of human GDF-9B. The human GDF-9B transcript can be detected only in the gonads by RT-PCR analysis, and in situ hybridization studies indicate that it is not expressed in small primary follicles but, rather, in the oocytes of late primary follicles. Functional studies using the Xenopus laeuis embryo model indicate that unlike the transforming growth factor-beta family members activin and bone morphogenetic protein-4, neither GDF-9 nor GDF-9B affects mesoderm induction, suggesting that they may use signaling pathways distinct from those well defined for activin and bone morphogenetic protein-4. We conclude that 1) both GDF-9 mRNA and protein are abundantly expressed in oocytes of primary follicles in human ovary, suggesting that the GDF-9 transcript is translated at this early stage of folliculogenesis; 2) human GDF-9B is specifically expressed in gonads at low levels; and 3) the expression of GDF-9 mRNA begins slightly earlier than that of GDF-9B in the human oocytes during follicular development. Our results are consistent with the suggestion that GDF-9 and GDF-9B may regulate human folliculogenesis in a manner specific to the ovary.

Characterization of the lysinuric protein intolerance (LPI) region within T-cell receptor alpha/delta gene cluster on chromosome site 14q11.

Lysinuric protein intolerance is a recessively inherited metabolic disease characterized by defective efflux of cationic amino acids at the basolateral membrane of the intestinal and renal tubular epithelium. Linkage analysis and further linkage disequilibrium in Finnish LPI families have earlier assigned LPI gene locus within or in close vicinity of T-cell receptor alpha/delta gene cluster on chromosome site 14q11. In the present work we have characterized the linkage defined LPI region using RH-mapping and fiber-FISH and searched the LPI gene from the reported sequence of the T-cell receptor gene.

Cloning of the APECED gene provides new insight into human autoimmunity.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is the only autoimmune disease characterized so far that is caused by a defect in a single gene. We have recently isolated the defective gene in this disease by positional cloning and have identified several different mutations in APECED patients. This novel gene, AIRE, contains two plant homeodomain (PHD)-type zinc finger motifs and a newly described putative DNA-binding domain SAND. We have further shown that the protein encoded by the AIRE gene is localized to the nuclear body-like structures of cell nuclei. Similar discrete speckles within the nucleus have been suggested to be involved in the regulation of transcription, oncogenesis and differentiation of cells. Together with the predicted structural features of the APECED protein the new data obtained both in vitro and ex vivo suggest that this protein participates in the regulation of gene expression in a restricted set of tissues and cells.

Localization of the APECED protein in distinct nuclear structures.

Autoimmune-polyendocrinopathy-candidiasis-ecto-dermaldystrophy (APECED) is the only systemic autoimmune disease with a monogenic background known so far revealing no association with the major histocompatibility complex region. We have recently isolated the gene defective in this syndrome and characterized several different mutations in individuals with the disorder. The novel gene, AIRE, contains a putative bipartite nuclear targeting signal predicting a nuclear location of the corresponding protein. The presence of two PHD-type zinc finger domains as well as the newly described putative DNA-binding domain, SAND, in the amino acid sequence of the APECED protein implies that it may be involved in the regulation of gene expression. Using transient expression of AIRE cDNA in mammalian cells we demonstrate here the nuclear location of the APECED protein. Immunohistochemical staining of transfected cells revealed that most of the recombinant 58 kDa APECED protein is present in the form of nuclear dots. By double immuno-fluorescence labelling we further show that these APECED-containing structures and the previously described PML nuclear bodies are largely non-overlapping. The AIRE protein was also visualized in multiple human tissues: a subset of the cells in thymus, in spleen and in lymph node showed nuclear staining with APECED antiserum. Immunofluorescence labelling of peripheral blood mononuclear leukocytes also revealed a nuclear body-like staining pattern in a fraction of these cells. These data from both in vitro and ex vivo systems, together with the predicted structural features of the APECED protein, suggest that this protein is most probably involved in the regulation of gene expression.

Mapping ESTs by fiber-FISH.

A visual transcript map of six genes was constructed on the chromosome 21q22.3 by high resolution fluorescence in situ hybridization (FISH). Expressed sequence tags (ESTs) from six genes-PWP2, KNP1, AIRE, C21orf3, SMT3A, and C21orf1-were successfully localized by fiber-FISH by use of sensitive tyramide-based detection. The sizes of the ESTs varied between 315 to 956 bp and most of them map within the 3'-untranslated region. The ESTs were assigned to and subsequently ordered within cosmid, PAC, and BAC clones hybridized on DNA fibers. Physical distances between ESTs and known markers were determined. Our results demonstrate the feasibility and accuracy of visual mapping EST sequences in relation to known markers. The main advantage of this approach is that it can be applied to finely map any of the database ESTs for positional cloning efforts. The sensitivity, specificity, and reproducibility of this high-resolution EST mapping technique is evaluated.