An autosomal locus causing autoimmune disease: autoimmune polyglandular disease type I assigned to chromosome 21.

Autoimmune polyglandular disease type I (APECED) is an autosomal recessive autoimmune disease characterized by a variable combination of the failure of the endocrine glands. The pathogenesis of this unique autoimmune disease is unknown; unlike many other autoimmune diseases, APECED does not show association to specific HLA haplotypes. Unravelling the APECED locus will identify a novel gene outside the HLA loci influencing the outcome of autoimmune diseases. We have assigned the disease locus to chromosome 21q22.3 by linkage analyses in 14 Finnish families. Linkage disequilibrium studies have significantly increased the informativeness of the analyses and helped to locate the critical DNA region for the APECED locus to just 500 kilobases, a much more precise definition than linkage analyses alone could achieve.

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.

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.

A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis.

Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member which is expressed in the oocytes in mouse ovaries (McGrath, S.A., Esquela, A.F., Lee, S.J., 1995. Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, 131-136). GDF-9 is indispensable for normal folliculogenesis since female mice deficient for the GDF-9 gene are infertile due to an arrest of follicular growth at the primary follicle stage (Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R. , Lu, N., Matzuk, M.M., 1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531-535). We searched the GenBank Expressed Sequence Tag (EST) database with the mouse GDF-9 cDNA sequence, and identified from a mouse 2-cell embryo library an EST cDNA that encodes a putative member of the TGF-b superfamily, and named it as GDF-9B. Northern blot hybridization analyses of mouse ovaries revealed a single transcript of approximately 4.0 kilobases (kb) for GDF-9B and of 2.0 kb for GDF-9. We cloned by reverse transcription-polymerase chain reaction from mouse ovarian RNA a partial 821-base pair GDF-9B cDNA that spans the sequence encoding the putative mature region of GDF-9B. The COOH-terminal region of GDF-9B appears to be 53% homologous to GDF-9. Moreover, like GDF-9, GDF-9B lacks the cysteine residue needed for the covalent dimerization of several TGF-b family members. Using in situ hybridization analysis, we demonstrate that GDF-9B and GDF-9 mRNAs are co-localized in the oocyte. We also show that GDF-9B and GDF-9 genes are co-ordinately expressed during follicular development.

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.

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.

Autoimmune polyglandular disease type I. Exclusion map using amplifiable multiallelic markers in a microtiter well format.

The pathogenetic background of human autoimmunity is only partially understood. By discovering the defective gene causing the autosomal recessive polyglandular autoimmune disease type I (PGD I, APECED) we hope to provide new insights into autoimmune responses in general. Here we have taken advantage of newly developed amplifiable multiallelic microsatellite markers and performed the analyses using the microtiter well format of the polymerase chain reaction. This rapid semiautomated protocol was applied to analyze 62 assigned highly polymorphic loci. The linkage analyses coupled with the EXCLUDE analysis resulted in an exclusion map of this polyglandular autoimmune disease and in the preliminary assignment of the APECED locus to chromosome 22. The method proved to be an effective and economical tool for gene mapping compared with standard blotting and hybridization.

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.

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.

Measurements of electromagnetic emissions from video display terminals at the frequency range from 30 Hz to 1 MHz.

Electric (E) and magnetic field (B) strengths or flux densities were measured at distances of 30 and 50 cm from the screen of video displays at a frequency range from 30 Hz to 1 MHz. The measurement system consisted of an optically coupled active dipole for E-fields, a magnetic field meter, a digital oscilloscope, a portable computer for data storage and a laboratory computer for Fourier analysis of the recorded signals. Comparison of measurement results with available broadband exposure standards or proposed standards indicated that magnetic flux density should be measured at both the (ELF) frequency range from 30 Hz to 300 Hz and at the (RF) frequency range from 10 kHz to 500 kHz. Alternatively, time derivative of magnetic flux density may be measured. Nor should the measurement of the electric field strength at the RF range be neglected. These conclusions, however, are valid only in relative terms. In all cases the exposure is at least one decade below the most stringent exposure limit. The maximum relative exposure 0.077 was obtained by applying the ACGIH standard for magnetic fields at a distance of 30 cm from the screen. The field strengths decrease by a factor varying from 2.5 to 3.5 at a distance of 50 cm, which is a more realistic distance when considering actual working conditions.

The shared image guiding the treatment process. A precondition for integration of the treatment of schizophrenia.

The aim of the study reported here was to develop psychotherapeutic in-patient treatment for acute schizophrenia, following the principles of a need-adapted approach. To improve the integration of experiences which hospital staff have with acutely psychotic patients and their families, systematic supervision sessions were organised. In these sessions, it was possible to achieve shared psychological images through while the whole staff could integrate patients' behaviour and symptoms, both symbolic and non-symbolic. Such an image was called 'the shared image guiding the treatment process' (SIGTP). The process of achieving the SIGTP was interpreted through Peircean semiotics, especially the concepts of indexical, iconic, and symbolical signs. An SIGTP was considered to have been achieved in the early phase of the supervision process in 32 of the 54 cases. For the patients, SIGTP and the need-adaptation connected with it meant achieving a more realistic and more functional ordering of their experiences.

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.

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.

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.

Messages from an isolate: lessons from the Finnish gene pool.

Genetic isolates are the result of some type of bottleneck in the history of a population, revealing the consequences of the founder effect and genetic drift on the population's gene pool. In human populations, isolation is suspected based on an exceptional geographic location or cultural history or on the prevalence of relatively rare genetic diseases. The concept of 'Finnish disease heritage' is well established in the literature, but solid data have only recently emerged regarding the uniformity of disease mutations at the molecular level in this population: for many Finnish diseases for which the molecular defect has been uncovered, over 90% of disease alleles carry the same causative mutation. This suggests dramatic isolation, especially in some subregions of the sparsely populated country. In Finland, this molecular information can be combined with the exceptional genealogical data offered by a well established church record system which dates back to 1640, containing detailed information on births, deaths, marriages and movements of the majority of the population. This provides excellent opportunities for special study designs for the identification not only of rare disease genes but also of major loci which contribute to complex diseases. The utilization of linkage disequilibrium and the search for shared haplotypes can be justified in subpopulations and patient materials from this genetic isolate. This review summarizes the current molecular evidence for genetic isolation as well as the utilization of some special strategies in the disease gene hunt in the Finnish population.

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.